cloning research paper

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Cloning articles from across Nature Portfolio

Cloning is a method that is used to produce genetically identical copies of pieces of DNA, cells or organisms. Cloning methods include: molecular cloning, which makes copies of pieces of DNA; cellular cloning, which makes copies of a cell; and whole organism cloning. All cloning methods involve DNA and cell manipulation.

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Cloning Fact Sheet

The term cloning describes a number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone. Researchers have cloned a wide range of biological materials, including genes, cells, tissues and even entire organisms, such as a sheep.

Do clones ever occur naturally?

Yes. In nature, some plants and single-celled organisms, such as bacteria , produce genetically identical offspring through a process called asexual reproduction. In asexual reproduction, a new individual is generated from a copy of a single cell from the parent organism.

Natural clones, also known as identical twins, occur in humans and other mammals. These twins are produced when a fertilized egg splits, creating two or more embryos that carry almost identical DNA . Identical twins have nearly the same genetic makeup as each other, but they are genetically different from either parent.

What are the types of artificial cloning?

There are three different types of artificial cloning: gene cloning, reproductive cloning and therapeutic cloning.

Gene cloning produces copies of genes or segments of DNA. Reproductive cloning produces copies of whole animals. Therapeutic cloning produces embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues.

Gene cloning, also known as DNA cloning, is a very different process from reproductive and therapeutic cloning. Reproductive and therapeutic cloning share many of the same techniques, but are done for different purposes.

What sort of cloning research is going on at NHGRI?

Gene cloning is the most common type of cloning done by researchers at NHGRI. NHGRI researchers have not cloned any mammals and NHGRI does not clone humans.

How are genes cloned?

Researchers routinely use cloning techniques to make copies of genes that they wish to study. The procedure consists of inserting a gene from one organism, often referred to as "foreign DNA," into the genetic material of a carrier called a vector. Examples of vectors include bacteria, yeast cells, viruses or plasmids, which are small DNA circles carried by bacteria. After the gene is inserted, the vector is placed in laboratory conditions that prompt it to multiply, resulting in the gene being copied many times over.

How are animals cloned?

In reproductive cloning, researchers remove a mature somatic cell , such as a skin cell, from an animal that they wish to copy. They then transfer the DNA of the donor animal's somatic cell into an egg cell, or oocyte, that has had its own DNA-containing nucleus removed.

Researchers can add the DNA from the somatic cell to the empty egg in two different ways. In the first method, they remove the DNA-containing nucleus of the somatic cell with a needle and inject it into the empty egg. In the second approach, they use an electrical current to fuse the entire somatic cell with the empty egg.

In both processes, the egg is allowed to develop into an early-stage embryo in the test-tube and then is implanted into the womb of an adult female animal.

Ultimately, the adult female gives birth to an animal that has the same genetic make up as the animal that donated the somatic cell. This young animal is referred to as a clone. Reproductive cloning may require the use of a surrogate mother to allow development of the cloned embryo, as was the case for the most famous cloned organism, Dolly the sheep.

What animals have been cloned?

Over the last 50 years, scientists have conducted cloning experiments in a wide range of animals using a variety of techniques. In 1979, researchers produced the first genetically identical mice by splitting mouse embryos in the test tube and then implanting the resulting embryos into the wombs of adult female mice. Shortly after that, researchers produced the first genetically identical cows, sheep and chickens by transferring the nucleus of a cell taken from an early embryo into an egg that had been emptied of its nucleus.

It was not until 1996, however, that researchers succeeded in cloning the first mammal from a mature (somatic) cell taken from an adult animal. After 276 attempts, Scottish researchers finally produced Dolly, the lamb from the udder cell of a 6-year-old sheep. Two years later, researchers in Japan cloned eight calves from a single cow, but only four survived.

Besides cattle and sheep, other mammals that have been cloned from somatic cells include: cat, deer, dog, horse, mule, ox, rabbit and rat. In addition, a rhesus monkey has been cloned by embryo splitting.

Have humans been cloned?

Despite several highly publicized claims, human cloning still appears to be fiction. There currently is no solid scientific evidence that anyone has cloned human embryos.

In 1998, scientists in South Korea claimed to have successfully cloned a human embryo, but said the experiment was interrupted very early when the clone was just a group of four cells. In 2002, Clonaid, part of a religious group that believes humans were created by extraterrestrials, held a news conference to announce the birth of what it claimed to be the first cloned human, a girl named Eve. However, despite repeated requests by the research community and the news media, Clonaid never provided any evidence to confirm the existence of this clone or the other 12 human clones it purportedly created.

In 2004, a group led by Woo-Suk Hwang of Seoul National University in South Korea published a paper in the journal Science in which it claimed to have created a cloned human embryo in a test tube. However, an independent scientific committee later found no proof to support the claim and, in January 2006, Science announced that Hwang's paper had been retracted.

From a technical perspective, cloning humans and other primates is more difficult than in other mammals. One reason is that two proteins essential to cell division, known as spindle proteins, are located very close to the chromosomes in primate eggs. Consequently, removal of the egg's nucleus to make room for the donor nucleus also removes the spindle proteins, interfering with cell division. In other mammals, such as cats, rabbits and mice, the two spindle proteins are spread throughout the egg. So, removal of the egg's nucleus does not result in loss of spindle proteins. In addition, some dyes and the ultraviolet light used to remove the egg's nucleus can damage the primate cell and prevent it from growing.

Do cloned animals always look identical?

No. Clones do not always look identical. Although clones share the same genetic material, the environment also plays a big role in how an organism turns out.

For example, the first cat to be cloned, named Cc, is a female calico cat that looks very different from her mother. The explanation for the difference is that the color and pattern of the coats of cats cannot be attributed exclusively to genes. A biological phenomenon involving inactivation of the X chromosome (See sex chromosome ) in every cell of the female cat (which has two X chromosomes) determines which coat color genes are switched off and which are switched on. The distribution of X inactivation, which seems to occur randomly, determines the appearance of the cat's coat.

What are the potential applications of cloned animals?

Reproductive cloning may enable researchers to make copies of animals with the potential benefits for the fields of medicine and agriculture.

For instance, the same Scottish researchers who cloned Dolly have cloned other sheep that have been genetically modified to produce milk that contains a human protein essential for blood clotting. The hope is that someday this protein can be purified from the milk and given to humans whose blood does not clot properly. Another possible use of cloned animals is for testing new drugs and treatment strategies. The great advantage of using cloned animals for drug testing is that they are all genetically identical, which means their responses to the drugs should be uniform rather than variable as seen in animals with different genetic make-ups.

After consulting with many independent scientists and experts in cloning, the U.S. Food and Drug Administration (FDA) decided in January 2008 that meat and milk from cloned animals, such as cattle, pigs and goats, are as safe as those from non-cloned animals. The FDA action means that researchers are now free to using cloning methods to make copies of animals with desirable agricultural traits, such as high milk production or lean meat. However, because cloning is still very expensive, it will likely take many years until food products from cloned animals actually appear in supermarkets.

Another application is to create clones to build populations of endangered, or possibly even extinct, species of animals. In 2001, researchers produced the first clone of an endangered species: a type of Asian ox known as a guar. Sadly, the baby guar, which had developed inside a surrogate cow mother, died just a few days after its birth. In 2003, another endangered type of ox, called the Banteg, was successfully cloned. Soon after, three African wildcats were cloned using frozen embryos as a source of DNA. Although some experts think cloning can save many species that would otherwise disappear, others argue that cloning produces a population of genetically identical individuals that lack the genetic variability necessary for species survival.

Some people also have expressed interest in having their deceased pets cloned in the hope of getting a similar animal to replace the dead one. But as shown by Cc the cloned cat, a clone may not turn out exactly like the original pet whose DNA was used to make the clone.

What are the potential drawbacks of cloning animals?

Reproductive cloning is a very inefficient technique and most cloned animal embryos cannot develop into healthy individuals. For instance, Dolly was the only clone to be born live out of a total of 277 cloned embryos. This very low efficiency, combined with safety concerns, presents a serious obstacle to the application of reproductive cloning.

Researchers have observed some adverse health effects in sheep and other mammals that have been cloned. These include an increase in birth size and a variety of defects in vital organs, such as the liver, brain and heart. Other consequences include premature aging and problems with the immune system. Another potential problem centers on the relative age of the cloned cell's chromosomes. As cells go through their normal rounds of division, the tips of the chromosomes, called telomeres, shrink. Over time, the telomeres become so short that the cell can no longer divide and, consequently, the cell dies. This is part of the natural aging process that seems to happen in all cell types. As a consequence, clones created from a cell taken from an adult might have chromosomes that are already shorter than normal, which may condemn the clones' cells to a shorter life span. Indeed, Dolly, who was cloned from the cell of a 6-year-old sheep, had chromosomes that were shorter than those of other sheep her age. Dolly died when she was six years old, about half the average sheep's 12-year lifespan.

What is therapeutic cloning?

Therapeutic cloning involves creating a cloned embryo for the sole purpose of producing embryonic stem cells with the same DNA as the donor cell. These stem cells can be used in experiments aimed at understanding disease and developing new treatments for disease. To date, there is no evidence that human embryos have been produced for therapeutic cloning.

The richest source of embryonic stem cells is tissue formed during the first five days after the egg has started to divide. At this stage of development, called the blastocyst, the embryo consists of a cluster of about 100 cells that can become any cell type. Stem cells are harvested from cloned embryos at this stage of development, resulting in destruction of the embryo while it is still in the test tube.

What are the potential applications of therapeutic cloning?

Researchers hope to use embryonic stem cells, which have the unique ability to generate virtually all types of cells in an organism, to grow healthy tissues in the laboratory that can be used replace injured or diseased tissues. In addition, it may be possible to learn more about the molecular causes of disease by studying embryonic stem cell lines from cloned embryos derived from the cells of animals or humans with different diseases. Finally, differentiated tissues derived from ES cells are excellent tools to test new therapeutic drugs.

What are the potential drawbacks of therapeutic cloning?

Many researchers think it is worthwhile to explore the use of embryonic stem cells as a path for treating human diseases. However, some experts are concerned about the striking similarities between stem cells and cancer cells. Both cell types have the ability to proliferate indefinitely and some studies show that after 60 cycles of cell division, stem cells can accumulate mutations that could lead to cancer. Therefore, the relationship between stem cells and cancer cells needs to be more clearly understood if stem cells are to be used to treat human disease.

What are some of the ethical issues related to cloning?

Gene cloning is a carefully regulated technique that is largely accepted today and used routinely in many labs worldwide. However, both reproductive and therapeutic cloning raise important ethical issues, especially as related to the potential use of these techniques in humans.

Reproductive cloning would present the potential of creating a human that is genetically identical to another person who has previously existed or who still exists. This may conflict with long-standing religious and societal values about human dignity, possibly infringing upon principles of individual freedom, identity and autonomy. However, some argue that reproductive cloning could help sterile couples fulfill their dream of parenthood. Others see human cloning as a way to avoid passing on a deleterious gene that runs in the family without having to undergo embryo screening or embryo selection.

Therapeutic cloning, while offering the potential for treating humans suffering from disease or injury, would require the destruction of human embryos in the test tube. Consequently, opponents argue that using this technique to collect embryonic stem cells is wrong, regardless of whether such cells are used to benefit sick or injured people.

Last updated: August 15, 2020

• Methodology
• Open access
• Published: 19 January 2015

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering

• Sayed Shahabuddin Hoseini 1 , 2 &
• Martin G Sauer 1 , 2 , 3  

Journal of Biological Engineering volume  9 , Article number:  2 ( 2015 ) Cite this article

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Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning.

Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells.

Conclusions

Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.

Various techniques were introduced for assembling new DNA sequences [ 1 – 3 ], yet the use of restriction endonuclease enzymes is the most widely used technique in molecular cloning. Whenever compatible restriction enzyme sites are available on both, insert and vector DNA sequences, cloning is straightforward; however, if restriction sites are incompatible or if there is even no restriction site available in the vicinity of the insert cassette, cloning might become more complex. The use of PCR primers, in which compatible restriction enzyme sites are embedded, can effectively solve this problem and facilitate multistep cloning procedures.

Although PCR cloning has been vastly used in biological engineering [ 4 – 8 ], practical guides explaining all necessary steps and tips in a consecutive order are scarce. Furthermore, the emergence of new high-fidelity DNA polymerases, kits, and powerful software makes the process of PCR cloning extremely fast and efficient. Here we sequentially explain PCR cloning from the analysis of the respective gene sequence, the design of PCR primers, performing the PCR procedure itself, sequencing the resulting PCR products, analysis of sequencing data, and finally the cloning of the PCR product into the final vector.

Results and discussion

Choosing proper restriction enzymes based on defined criteria.

In order to proceed with a concise example, tdTomato fluorescent protein was cloned into an alpharetroviral vector. Consecutively, a murine leukemia cell line expressing tdTomato was generated. This cell line will be used to track tumor cells upon injection into mice in preclinical immunotherapy studies. However, this cloning method is applicable to any other gene. To begin the cloning project, the gene of interest (GOI) should be analyzed. First, we check whether our annotated sequence has a start codon (ATG, the most common start codon) and one of the three stop codons (TAA, TAG, TGA). In case the gene was previously manipulated or fused to another gene (e.g. via a 2A sequence), it happens that a gene of interest might not have a stop codon [ 9 ]. In such cases, a stop codon needs to be added to the end of your annotated sequence. It is also beneficial to investigate whether your GOI contains an open reading frame (ORF). This is important since frequent manipulation of sequences either by software or via cloning might erroneously add or delete nucleotides. We use Clone Manager software (SciEd) to find ORFs in our plasmid sequences; however, there are several free websites you can use to find ORFs including the NCBI open reading frame finder ( http://www.ncbi.nlm.nih.gov/gorf/gorf.html ).

The tdTomato gene contains ATG start codon and TAA stop codon (Figure  1 ). The size of the tdTomato gene is 716 bp.

Overview of the start and the end of the gene of interest. (A) The nucleotide sequences at the start and the end of the tdTomato gene are shown. The coding strand nucleotides are specified in bold (B) The nucleotide sequences of the forward and reverse primers containing proper restriction enzyme sites and the Kozak sequence are shown.

In a next step, PCR primers that include proper restriction enzyme sites need to be designed for the amplification of the GOI. Several criteria should be considered in order to choose the optimal restriction enzymes. First, binding sites for restriction enzymes should be ideally available at a multiple cloning site within the vector. Alternatively they can be located downstream of the promoter in your vector sequence. Restriction enzymes should be single cutters (single cutters target one restriction site only within a DNA sequence) (Figure  2 A). If they are double or multiple cutters, they should cut within a sequence that is not necessary for proper functioning of the vector plasmid and will finally be removed (Figure  2 B). It is also possible to choose one double cutter or multiple cutter enzymes cutting the vector downstream of the promoter and also not within a vital sequence of the plasmid (Figure  2 C). Double cutter or multiple cutter enzymes have two or more restriction sites on a DNA sequence, respectively. Cutting the vector with double or multiple cutters would give rise to two identical ends. In such a case, the insert cassette should also contain the same restriction enzyme sites on both of its ends. Therefore, when the insert and vector fragments are mixed in a ligation experiment, the insert can fuse to the vector in either the right orientation (from start codon to stop codon) or reversely (from stop codon to start codon). A third scenario can occur, if the vector fragment forms a self-ligating circle omitting the insert at all. Once the DNA has been incubated with restriction enzymes, dephosphorylation of the 5′ and 3′ ends of the vector plasmid using an alkaline phosphatase enzyme will greatly reduce the risk of self-ligation [ 10 ]. It is therefore important to screen a cloning product for those three products (right orientation, reverse orientation, self-ligation) after fragment ligation.

Choosing proper restriction enzymes based on defined criteria for PCR cloning. (A) Two single-cutter restriction enzymes (E1 and E2) are located downstream of the promoter. (B) E1 and E2 restriction enzymes cut the plasmid downstream of the promoter several (here two times for each enzyme) times. (C) The E1 restriction enzyme cuts the plasmid downstream of the promoter more than once. (D) The PCR product, which contains the tdTomato gene and the restriction enzyme sites, was run on a gel before being extracted for downstream applications.

Second, due to higher cloning efficiency using sticky-end DNA fragments, it is desirable that at least one (better both) of the restriction enzymes is a so-called sticky-end cutter. Sticky end cutters cleave DNA asymmetrically generating complementary cohesive ends. In contrast, blunt end cutters cut the sequence symmetrically leaving no overhangs. Cloning blunt-end fragments is more difficult. Nevertheless, choosing a higher insert/vector molar ratio (5 or more) and the use 10% polyethylene glycol (PEG) can improve ligation of blunt-end fragments [ 11 ].

Third, some restriction enzymes do not cut methylated DNA. Most of the strains of E. coli contain Dam or Dcm methylases that methylate DNA sequences. This makes them resistant to methylation-sensitive restriction enzymes [ 12 ]. Since vector DNA is mostly prepared in E. coli , it will be methylated. Therefore avoiding methylation-sensitive restriction enzymes is desirable; however, sometimes the isoschizomer of a methylation-sensitive restriction enzyme is resistant to methylation. For example, the Acc 65I enzyme is sensitive while its isoschizomer kpn I is resistant to methylation [ 13 ]. Isoschizomers are restriction enzymes that recognize the same nucleotide sequences. If there remains no other option than using methylation-sensitive restriction enzymes, the vector DNA needs to be prepared in dam − dcm − E. coli strains. A list of these strains and also common E. coli host strains for molecular cloning is summarized in Table  1 . Information regarding the methylation sensitivity of restriction enzymes is usually provided by the manufacturer.

Fourth, it makes cloning easier if the buffer necessary for the full functionality of restriction enzymes is the same because one can perform double restriction digest. This saves time and reduces the DNA loss during purification. It may happen that one of the restriction enzymes is active in one buffer and the second enzyme is active in twice the concentration of the same buffer. For example the Nhe I enzyme from Thermo Scientific is active in Tango 1X buffer (Thermo Scientific) and Eco R1 enzyme is active in Tango 2X buffer (Thermo Scientific). In such cases, the plasmid DNA needs to be first digested by the enzyme requiring the higher buffer concentration (here Eco R1). This will be followed by diluting the buffer for the next enzyme (requiring a lower concentration (here Nhe I)) in the same buffer. However, the emergence of universal buffers has simplified the double digest of DNA sequences [ 15 ]. In our example the vector contains the Age I and Sal I restriction sites. These enzyme sites were used for designing PCR primers (Figure  1 ). It is essential for proper restriction enzyme digestion that the plasmid purity is high. DNA absorbance as measured by a spectrophotometer can be used to determine the purity after purification. DNA, proteins, and solvents absorb at 260 nm, 280 nm, and 230 nm, respectively. An OD 260/280 ratio of >1.8 and an OD 260/230 ratio of 2 to 2.2 is considered to be pure for DNA samples [ 16 ]. The OD 260/280 and 260/230 ratios of our exemplary plasmid preparations were 1.89 and 2.22, respectively. We observed that the purity of the gel-extracted vector and insert DNA fragments were lower after restriction digest; ligation works even in such cases, however, better results can be expected using high-purity fragments.

The following plasmid repository website can be useful for the selection of different vectors (viral expression and packaging, empty backbones, fluorescent proteins, inducible vectors, epitope tags, fusion proteins, reporter genes, species-specific expression systems, selection markers, promoters, shRNA expression and genome engineering): http://www.addgene.org/browse/ .

A collection of cloning vectors of E. coli is available under the following website: http://www.shigen.nig.ac.jp/ecoli/strain/cvector/cvectorExplanation.jsp .

Designing cloning primers based on defined criteria

For PCR primer design, check the start and stop codons of your GOI. Find the sequence of the desired restriction enzymes (available on the manufacturers’ websites) for the forward primer (Figure  3 A). It needs to be located before the GOI (Figure  1 B). The so-called Kozak sequence is found in eukaryotic mRNAs and improves the initiation of translation [ 17 ]. It is beneficial to add the Kozak sequence (GCCACC) before the ATG start codon since it increases translation and expression of the protein of interest in eukaryotes [ 18 ]. Therefore, we inserted GCCACC immediately after the restriction enzyme sequence Age I and before the ATG start codon. Then, the first 18 to 30 nucleotides of the GOI starting from the ATG start codon are added to the forward primer sequence. These overlapping nucleotides binding to the template DNA determine the annealing temperature (Tm). The latter is usually higher than 60°C. Here, we use Phusion high-fidelity DNA polymerase (Thermo Scientific). You can use the following websites for determination of the optimal Tm: http://www.thermoscientificbio.com/webtools/tmc/ .

Designing primers based on defined criteria for PCR cloning. (A-B) Sequences of the forward and the reverse primer are depicted. The end of the coding strand is to be converted into the reverse complement format for the reverse primer design. For more information, please see the text.

https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator .

The Tm of our forward primer is 66°C.

Choose the last 18 to 30 nucleotides including the stop codon of your GOI for designing the reverse primer (Figure  3 B). Then calculate the Tm for this sequence which should be above 60°C and close to the Tm of the forward primer. Tm of the overlapping sequence of our reverse primer was 68°C. Then, add the target sequence of the second restriction enzyme site (in this case Sal I) immediately after the stop codon. Finally, convert this assembled sequence to a reverse-complement sequence. The following websites can be used to determine the sequence of the reverse primer:

http://reverse-complement.com/

http://www.bioinformatics.org/sms/rev_comp.html This is important since the reverse primer binds the coding strand and therefore its sequence (5′ → 3′) must be reverse-complementary to the sequence of the coding strand (Figure  1 A).

Performing PCR using proofreading polymerases

Since the PCR reaction follows logarithmic amplification of the target sequence, any replication error during this process will be amplified. The error rate of non-proofreading DNA polymerases, such as the Taq polymerase, is about 8 × 10 −6 errors/bp/PCR cycle [ 19 ]; however, proofreading enzymes such as Phusion polymerase have a reported error rate of 4.4 × 10 −7 errors/bp/PCR cycle. Due to its superior fidelity and processivity [ 20 – 22 ], the Phusion DNA polymerase was used in this example. It should be noted that Phusion has different temperature requirements than other DNA polymerases. The primer Tm for Phusion is calculated based on the Breslauer method [ 23 ] and is higher than the Tm using Taq or pfu polymerases. To have optimal results, the Tm should be calculated based on information found on the website of the enzyme providers. Furthermore, due to the higher speed of Phusion, 15 to 30 seconds are usually enough for the amplification of each kb of the sequence of interest.

After the PCR, the product needs to be loaded on a gel (Figure  2 D). The corresponding band needs to be cut and the DNA extracted. It is essential to sequence the PCR product since the PCR product might include mutations. There are several PCR cloning kits available some of which are shown in Table  2 . We used the pJET1.2/blunt cloning vector (Thermo Scientific, patent publication: US 2009/0042249 A1, Genbank accession number EF694056.1) and cloned the PCR product into the linearized vector. This vector contains a lethal gene ( eco47IR ) that is activated in case the vector becomes circularized. However, if the PCR product is cloned into the cloning site within the lethal gene, the latter is disrupted allowing bacteria to grow colonies upon transformation. Circularized vectors not containing the PCR product express the toxic gene, which therefore kills bacteria precluding the formation of colonies. Bacterial clones are then to be cultured, plasmid DNA consecutively isolated and sequenced. The quality of isolated plasmid is essential for optimal sequencing results. We isolated the plasmid DNA from a total of 1.5 ml cultured bacteria (yield 6 μg DNA; OD 260/280 = 1.86; OD 260/230 = 2.17) using a plasmid mini-preparation kit (QIAGEN). The whole process of PCR, including cloning of the PCR product into the sequencing vector and transfection of bacteria with the sequencing vector can be done in one day. The next day, bacterial clones will be cultured overnight before being sent for sequencing.

Analysis of sequencing data

Sequencing companies normally report sequencing data as a FASTA file and also as ready nucleotide sequences via email. For sequence analysis, the following websites can be used:

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://xylian.igh.cnrs.fr/bin/align-guess.cgi

Here we will focus on the first website. On this website page, click on the “nucleotide blast” option (Figure  4 A). A new window opens. By default, the “blastn” (blast nucleotide sequences) option is marked (Figure  4 B). Then check the box behind “Align two or more sequences”. Now two boxes will appear. In the “Enter Query Sequence” box (the upper box), insert the desired sequence of your gene of interest, which is flanked by the restriction sites you have already designed for your PCR primers. In the “Enter Subject Sequence” box (the lower box), enter the sequence or upload the FASTA file you have received from the sequencing company. Then click the “BLAST” button at the bottom of the page. After a couple of seconds, the results will be shown on another page. A part of the alignment data is shown in Figure  4 C. For interpretation, the following points should be considered: 1) the number of identical nucleotides (shown under the “Identities” item) must be equal to the nucleotide number of your gene of interest. In our example, the number of nucleotides of the tdTomato gene together with those of the restriction enzyme sites and the Kozak sequence was 735. This equals the reported number (Figure  4 C). 2) The sequence identity (under the “Identities” item) should be 100%. Occasionally, the sequence identity is 100% but the number of identical nucleotides is lower than expected. This can happen if one or more of the initial nucleotides are absent. Remember, all sequencing technologies have an error rate. For Sanger sequencing, this error rate is reported to range from 0.001% to 1% [ 30 – 33 ]. Nucleotide substitution, deletion or insertion can be identified by analyzing the sequencing results [ 34 ]. Therefore, if the sequence identity does not reach 100%, the plasmid should be resequenced in order to differentiate errors of the PCR from simple sequencing errors. 3) Gaps (under the “Gaps” item) should not be present. If gaps occur, the plasmid should be resequenced.

Sequence analysis of the PCR product using the NCBI BLAST platform. (A) On the NCBI BLAST webpage, the “nucleotide blast” option is chosen (marked by the oval line). (B) The “blastn” option appears by default (marked by the circle). The sequence of the gene of interest (flanked by the restriction sites as previously designed for the PCR primers) and the PCR product are to be inserted to the “Enter Query Sequence” and “Enter Subject Sequence” boxes. Sequences can also be uploaded as FASTA files. (C) Nucleotide alignment of the first 60 nucleotides is shown. Two important items for sequence analysis are marked by oval lines.

The average length of a read, or read length, is at least 800 to 900 nucleotides for Sanger sequencing [ 35 ]. For the pJET vector one forward and one reverse primer need to be used for sequencing the complete gene. These primers can normally cover a gene size ranging up to 1800 bp. If the size of a gene is larger than 1800, an extra primer should be designed for each 800 extra nucleotides. Since reliable base calling does not start immediately after the primer, but about 45 to 55 nucleotides downstream of the primer [ 36 ], the next forward primer should be designed to start after about 700 nucleotides from the beginning of the gene. Different websites, including the following, can be used to design these primers:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.yeastgenome.org/cgi-bin/web-primer

http://www.genscript.com/cgi-bin/tools/sequencing_primer_design

Being 735 bp in length, the size of the PCR product in this example was well within the range of the pJET sequencing primers.

After choosing the sequence-verified clone, vector and insert plasmids were digested by the Age I and Sal I restriction enzymes (Figure  5 ). This was followed by gel purification and ligation of the fragments. Transformation of competent E. coli with the ligation mixture yielded several clones that were screened by restriction enzymes. We assessed eight clones, all of which contained the tdTomato insert (Figure  6 ). It is important to pick clones that are large. Satellite clones might not have the right construct. We used a fast plasmid mini-preparation kit (Zymo Research) to extract the plasmid from 0.6 ml bacterial suspension. The yield and purity were satisfying for restriction enzyme-based screening (2.3 μg DNA; OD 260/280 = 1.82; OD 260/230 = 1.41). For large-scale plasmid purification, a maxi-preparation kit (QIAGEN) was used to extract the plasmid from 450 ml of bacterial culture (yield 787 μg DNA; OD 260/280 = 1.89; OD 260/230 = 2.22). The expected yield of a pBR322-derived plasmid isolation from 1.5 ml and 500 ml bacterial culture is about 2-5 μg and 500-4000 μg of DNA, respectively [ 37 ].

Vector and insert plasmid maps A) Illustration of the CloneJET plasmid containing the PCR product. Insertion of the PCR product in the cloning site of the plasmid disrupts the integrity of the toxic gene eco47IR and allows the growth of transgene positive clones. The plasmid was cut with the Age I and Sal I enzymes generating two fragments of 3 kb and 0.7 kb in size. The 0.7 kb fragment (tdTomato gene) was used as the insert for cloning. (B) Illustration of the vector plasmid. The plasmid was cut with the Age I and Sal I enzymes generating two fragments of 4.9 kb and 0.7 kb in size. The 4.9 kb fragment was used as the vector for cloning. AMP: Ampicillin resistance gene; PRE: posttranscriptional regulatory element; MPSV: myeloproliferative sarcoma virus promoter.

Screening of the final plasmid with restriction enzymes. Illustration of the final plasmid is shown. For screening, the plasmid was cut with the Bsiw I enzyme generating two fragments of 4.8 kb and 0.8 kb in size. AMP: Ampicillin resistance gene; PRE: posttranscriptional regulatory element; MPSV: myeloproliferative sarcoma virus promoter.

Some plasmids tend to recombine inside the bacterial host creating insertions, deletions and recombinations [ 38 ]. In these cases, using a recA-deficient E. coli can be useful (Table  1 ). Furthermore, if the GOI is toxic, incubation of bacteria at lower temperatures (25-30°C) and using ABLE C or ABLE K strains might circumvent the problem.

Viral production and transduction of target cells

To investigate the in vitro expression of the cloned gene, HEK293T cells were transfected with plasmids encoding the tdTomato gene, alpharetroviral Gag/Pol, and the vesicular stomatitis virus glycoprotein (VSVG) envelope. These cells, which are derived from human embryonic kidney, are easily cultured and readily transfected [ 39 ]. Therefore they are extensively used in biotechnology and gene therapy to generate viral particles. HEK293T cells require splitting every other day using warm medium. They should not reach 100% confluency for optimal results. To have good transfection efficiency, these cells need to be cultured for at least one week to have them in log phase. Transfection efficiency was 22%, as determined based on the expression of tdTomato by fluorescence microscopy 24 hours later (Figure  7 A-B). To generate a murine leukemia cell line expressing the tdTomato gene for immunotherapy studies, C1498 leukemic cells were transduced with freshly harvested virus (36 hours of transfection). Imaging studies (Figure  7 C) and flow cytometric analysis (Figure  7 D) four days after transduction confirmed the expression of tdTomato in the majority of the cells.

Assessing in vitro expression of the cloned gene. (A, B) HEK293T cells were transfected with Gag/Pol, VSVG, and tdTomato plasmids. The expression of the tdTomato gene was assessed using a fluorescence microscope. Fluorescent images were superimposed on a bright-field image for the differentiation of positively transduced cells. Transfection efficiency was determined based on the expression of tdTomato after 24 hours. Non-transfected HEK293T cells were used as controls (blue histogram). (C, D) The murine leukemia cell line C1498 was transduced with fresh virus. Four days later, transgene expression was assessed by fluorescence microscopy (C) and flow cytometry (D) . Non-transduced C1498 cells were used as controls (blue histogram). Scale bars represent 30 μm.

In this manuscript, we describe a simple and step-by-step protocol explaining how to exploit the power of PCR to clone a GOI into a vector for genetic engineering. Several PCR-based creative methods have been developed being extremely helpful for the generation of new nucleotide sequences. This includes equimolar expression of several proteins by linking their genes via a self-cleaving 2A sequence [ 40 , 41 ], engineering fusion proteins, as well as the use of linkers for the design of chimeric proteins [ 42 – 44 ]. Furthermore, protein tags [ 45 , 46 ] and mutagenesis (site-directed, deletions, insertions) [ 47 ] have widened the applications of biological engineering. The protocol explained in this manuscript covers for most situations of PCR-assisted cloning; however, alternative PCR-based methods are available being restriction enzyme and ligation independent [ 6 , 48 – 51 ]. They are of special interest in applications where restriction enzyme sites are lacking; nevertheless, these methods might need several rounds of PCR or occasionally a whole plasmid needs to be amplified. In such cases, the chance of PCR errors increases and necessitates sequencing of multiple clones. In conclusion, this guideline assembles a simple and straightforward protocol using resources that are tedious to collect on an individual basis thereby trying to minimize errors and pitfalls from the beginning.

Cell lines and media

The E. coli HB101 was used for the preparation of plasmid DNA. The bacteria were cultured in Luria-Bertani (LB) media. Human embryonic kidney (HEK) 293 T cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 mg/ml streptomycin, and 100 units/ml penicillin. A myeloid leukemia cell line C1498 [ 52 ], was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with the same reagents used for DMEM. Cells were split every other day to keep them on log phase.

Plasmids, primers, PCR and sequencing

A plasmid containing the coding sequence of the tdTomato gene, plasmid containing an alpha-retroviral vector, and plasmids containing codon-optimized alpharetroviral gag/pol [ 53 ] were kindly provided by Axel Schambach (MHH Hannover, Germany). A forward (5′- ACCGGTGCCACCATGGCCACAACCATGGTG-3′) and a reverse (5′-GTCGACTTACTTGTACAGCTCGTCCATGCC-3′) primer used for the amplification of the tdTomato gene were synthesized by Eurofins Genomics (Ebersberg, Germany).

The optimal buffers for enzymes or other reagents were provided by the manufacturers along with the corresponding enzymes or inside the kits. If available by the manufacturers, the pH and ingredients of buffers are mentioned. Primers were dissolved in ultrapure water at a stock concentration of 20 pmol/μl. The template plasmid was diluted in water at a stock concentration of 50 ng/μl. For PCR, the following reagents were mixed and filled up with water to a total volume of 50 μl: 1 μl plasmid DNA (1 ng/μl final concentration), 1.25 μl of each primer (0.5 pmol/μl final concentration for each primer), 1 μL dNTP (10 mM each), 10 μl of 5X Phusion HF buffer (1X buffer provides 1.5 mM MgCl2), and 0.5 μl Phusion DNA polymerase (2U/μl, Thermo Scientific).

PCR was performed using a peqSTAR thermocycler (PEQLAB Biotechnologie) at: 98°C for 3 minutes; 25 cycles at 98°C for 10 seconds, 66°C for 30 seconds, 72°C for 30 seconds; and 72°C for 10 minutes. To prepare a 0.8% agarose gel, 0.96 g agarose (CARL ROTH) was dissolved in 120 ml 1X TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH of 50X TAE: 8.4) and boiled for 4 minutes. Then 3 μl SafeView nucleic acid stain (NBS Biologicals) was added to the solution and the mixture was poured into a gel-casting tray.

DNA was mixed with 10 μl loading dye (6X) (Thermo Scientific) and loaded on the agarose gel (CARL ROTH) using 80 V for one hour in TAE buffer. The separated DNA fragments were visualized using an UV transilluminator (365 nm) and quickly cut to minimize the UV exposure. DNA was extracted from the gel slice using Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The concentration of DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

For sequence validation, the PCR product was subcloned using CloneJET PCR cloning kit (Thermo Scientific). 1 μl of blunt vector (50 ng/μl), 50 ng/μl of the PCR product, and 10 μl of 2X reaction buffer (provided in the kit) were mixed and filled with water to a total volume of 20 μl. 1 μl of T4 DNA ligase (5 U/μl) was added to the mixture, mixed and incubated at room temperature for 30 minutes. For bacterial transfection, 10 μl of the mixture was mixed with 100 μl of HB101 E. coli competent cells and incubated on ice for 45 minutes. Then the mixture was heat-shocked (42°C/2 minutes), put on ice again (5 minutes), filled up with 1 ml LB medium and incubated in a thermomixer (Eppendorf) for 45 minutes/37°C/450RPM. Then the bacteria were spun down for 4 minutes. The pellet was cultured overnight at 37°C on an agarose Petri dish containing 100 μg/mL of Ampicillin. The day after, colonies were picked and cultured overnight in 3 ml LB containing 100 μg/mL of ampicillin.

After 16 hours (overnight), the plasmid was isolated from the cultured bacteria using the QIAprep spin miniprep kit (QIAGEN) according to the manufacturer’s instructions. 720 to 1200 ng of plasmid DNA in a total of 12 μl water were sent for sequencing (Seqlab) in Eppendorf tubes. The sequencing primers pJET1.2-forward (5′-CGACTCACTATAGGGAG-3′), and pJET1.2-reverse (5′-ATCGATTTTCCATGGCAG-3′), were generated by the Seqlab Company (Göttingen, Germany). An ABI 3730XL DNA analyzer was used by the Seqlab Company to sequence the plasmids applying the Sanger method. Sequence results were analyzed using NCBI Blast as explained in the Results and discussion section.

Manipulation of DNA fragments

For viewing plasmid maps, Clone Manager suite 6 software (SciEd) was used. Restriction endonuclease enzymes (Thermo Scientific) were used to cut plasmid DNA. 5 μg plasmid DNA, 2 μl buffer O (50 mM Tris–HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 0.1 mg/mL BSA, Thermo Scientific), 1 μl Sal I (10 U), and 1 μl AgeI (10 U) were mixed in a total of 20 μl water and incubated (37°C) overnight in an incubator to prevent evaporation and condensation of water under the tube lid. The next day, DNA was mixed with 4 μl loading dye (6X) (Thermo Scientific) and run on a 0.8% agarose gel at 80 V for one hour in TAE buffer. The agarose gel (120 ml) contained 3 μl SafeView nucleic acid stain (NBS Biologicals). The bands were visualized on a UV transilluminator (PEQLAB), using a wavelength of 365 nm, and quickly cut to minimize the UV damage. DNA was extracted from the gel slices using the Zymoclean™ gel DNA recovery kit (Zymo Research). The concentration of DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

For the ligation of vector and insert fragments, a ligation calculator was designed (the Excel file available in the Additional file 1 ) for easy calculation of the required insert and vector volumes. The mathematical basis of the calculator is inserted into the excel spreadsheet. The size and concentration of the vector and insert fragments and the molar ratio of vector/insert (normally 1:3) must be provided for the calculation. Calculated amounts of insert (tdTomato) and vector (alpha-retroviral backbone) were mixed with 2 μl of 10X T4 ligase buffer (400 mM Tris–HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C), Thermo Scientific), 1 μl of T4 ligase (5 U/μl, Thermo Scientific), filled up to 20 μl using ultrapure water and incubated overnight at 16°C. The day after, HB101 E. coli was transfected with the ligation mixture as mentioned above. The clones were picked and consecutively cultured for one day in LB medium containing ampicillin. Plasmid DNA was isolated using Zyppy™ plasmid miniprep kit (Zymo Research) and digested with proper restriction enzymes for screening. Digested plasmids were mixed with the loading dye and run on an agarose gel as mentioned above. The separated DNA fragments were visualized using a Gel Doc™ XR+ System (BIO-RAD) and analyzed by the Image Lab™ software (BIO-RAD). The positive clone was cultured overnight in 450 ml LB medium containing ampicillin. Plasmid DNA was isolated using QIAGEN plasmid maxi kit (QIAGEN), diluted in ultrapure water and stored at −20°C for later use.

Production of viral supernatant and transduction of cells

HEK293T cells were thawed, split every other day for one week and grown in log phase. The day before transfection, 3.5 × 10 6 cells were seeded into tissue culture dishes (60.1 cm 2 growth surface, TPP). The day after, the cells use to reach about 80% confluence. If over confluent, transfection efficiency decreases. The following plasmids were mixed in a total volume of 450 μl ultrapure water: codon-optimized alpharetroviral gag/pol (2.5 μg), VSVG envelope (1.5 μg), and the alpharetroviral vector containing the tdTomato gene (5 μg). Transfection was performed using calcium phosphate transfection kit (Sigma-Aldrich). 50 μl of 2.5 M CaCl 2 was added to the plasmid DNA and the mixture was briefly vortexed. Then, 0.5 ml of 2X HEPES buffered saline (provided in the kit) was added to a 15 ml conical tube and the calcium-DNA mixture was added dropwise via air bubbling and incubated for 20 minutes at room temperature. The medium of the HEK293T cells was first replaced with 8 ml fresh medium (DMEM containing FCS and supplement as mentioned above) containing 25 μM chloroquine. Consecutively the transfection mixture was added. Plates were gently swirled and incubated at 37°C. After 12 hours, the medium was replaced with 6 ml of fresh RPMI containing 10% FCS and supplements. Virus was harvested 36 hours after transfection, passed through a Millex-GP filter with 0.22 μm pore size (Millipore), and used freshly to transduce C1498 cells. Before transduction, 24 well plates were coated with retronectin (Takara, 280 μl/well) for 2 hours at room temperature. Then, retronectin was removed and frozen for later use (it can be re-used at least five times) and 300 μl of PBS containing 2.5% bovine serum albumin (BSA) was added to the wells for 30 minutes at room temperature. To transduce C1498 cells, 5 × 10 4 of cells were spun down and resuspended with 1 ml of fresh virus supernatant containing 4 μg/ml protamine sulfate. The BSA solution was removed from the prepared plates and plates were washed two times with 0.5 ml PBS. Then cells were added to the wells. Plates were centrifuged at 2000RPM/32°C/90 minutes. Fresh medium was added to the cells the day after.

Flow cytometry and fluorescence microscope

For flow cytometry assessment, cells were resuspended in PBS containing 0.5% BSA and 2 mM EDTA and were acquired by a BD FACSCanto™ (BD Biosciences) flow cytometer. Flow cytometry data were analyzed using FlowJo software (Tree Star). Imaging was performed with an Olympus IX71 fluorescent microscope equipped with a DP71 camera (Olympus). Images were analyzed with AxioVision software (Zeiss). Fluorescent images were superimposed on bright-field images using adobe Photoshop CS4 software (Adobe).

Abbreviations

Polymerase chain reaction

Gene of interest

Open reading frame

Melting temperature

Basic local alignment search tool

Vesicular stomatitis virus G glycoprotein

Luria-Bertani

Dulbecco’s Modified Eagle medium

Roswell Park Memorial Institute

Bovine serum albumin

Ethylenediaminetetraacetic acid

Fluorescence-activated cell sorting

Human embryonic kidney

Phosphate buffered saline

Fetal calf serum

Hydroxyethyl-piperazineethane-sulfonic acid

Ampicillin resistance gene

Posttranscriptional regulatory element

Myeloproliferative sarcoma virus promoter.

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Acknowledgments

The authors would like to thank Jessica Herbst, Abbas Behpajooh, Christian Kardinal and Juwita hübner for their fruitful discussions. We also thank Gang Xu for helping to design the cover page. This work was supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, the Deutsche Jose-Carreras Leukämiestiftung (grants SFB-738, IFB-TX CBT_6, DJCLS R 14/10 to M.G.S.) and the Ph.D. program Molecular Medicine of the Hannover Medical School.

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Sayed Shahabuddin Hoseini & Martin G Sauer

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The authors declare that they have no competing interests.

Authors’ contributions

SSH conceived the study subject, carried out experiments and drafted the initial manuscript. MGS participated in study design and coordination and edited the manuscript. Both authors have read and approved the final manuscript.

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13036_2014_161_moesm1_esm.xlsx.

Additional file 1: Ligation calculator. To calculate the amounts of the vector and insert fragments for a ligation reaction, you need to provide the size of the vector and insert (in base pairs), the molar ration of insert/vector (normally 3 to 5), vector amount (normally 50 to 100 ng), and vector and insert fragment concentrations (ng/μl). The computational basis of this ligation calculator is mentioned in the lower box. (XLSX 50 KB)

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Hoseini, S.S., Sauer, M.G. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. J Biol Eng 9 , 2 (2015). https://doi.org/10.1186/1754-1611-9-2

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Received : 05 September 2014

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• Saudi J Biol Sci
• v.28(5); 2021 May

Animal cloning and consumption of its by-products: A scientific and Islamic perspectives

Mohd izhar ariff mohd kashim.

a Center of Shariah, Faculty of Islamic Studies, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

b Institute of Islam Hadhari, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

Nur Asmadayana Hasim

Diani mardiana mat zin.

c PERMATA Insan College, Universiti Sains Islam Malaysia, Bandar Baru Nilai, 71800 Nilai, Negeri Sembilan, Malaysia

Latifah Amin

d Pusat Citra Universiti, Universiti Kebangsaan Malaysia, 43600 Bandar Baru Bangi, Malaysia

Mohd Helmy Mokhtar

e Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia

Safiyyah Shahimi

f Department of Food Science, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

Sahilah Abd. Mutalib

Islam is a religion that inspires its followers to seek knowledge continually and nurtures innovation, within the realms of Islamic rulings, towards an ameliorated quality of life. Up-to-date biotechnological techniques, specifically animal cloning, are involved in advancing society’s health, social, and economic domains. The goal of animal cloning includes the production of genetically modified animal for human consumption. Therefore, this research endeavoured to study animal cloning’s current scientific findings, examine the by-product of said process, and determine its permissibility in an Islamic context. This study employed descriptive literature reviews. Results concluded that animal cloning, especially in mammals, does not occur naturally as in plants. A broadly trusted and efficient animal cloning method is known as Somatic Cell Nuclear Transfer (SCNT), which includes three principal steps: oocyte enucleation; implantation of donor cells (or nucleus); and the activation of the embryo. Nevertheless, the limitations of SCNT, particularly to the Large Offspring Syndrome (LOS), should be noted. One of the forms of the application of animal cloning is in agriculture. From an Islamic perspective, determining the permissibility of consuming cloned animals as food is essentially based on whether the cloned animal conforms to Islamic law’s principles and criteria. Islam interdicts animal cloning when it is executed without benefiting humans, religion, or society. Nonetheless, if it is done to preserve the livelihood and the needs of a community, then the process is deemed necessary and should be administered following the conditions outlined in Islam. Hence, the Islamic ruling for animal cloning is not rigid and varies proportionately with the current fatwa.

1. Introduction

Islam urges one to seek knowledge and innovation to improve overall well-being and quality of life. Nevertheless, in recognising the sense of enhancing the quality of life, one must also ensure that their efforts comply with the Islamic rulings and not trigger adverse consequences, especially to humans. Modern biotechnology constitutes one of the key focuses of research in the last three decades. Since its conceptualisation, cloning has been a well-debated subject in addressing modern biotechnology issues in both the public and scientific fields ( Larijani and Zahedi, 2004 ). The swift scientific advancement of animal cloning has garnered considerable attention, which led to critical consideration and review of the process ( Fiester, 2005 , Zin et al., 2019 ). Modern biotechnology applications can be broadly identified in genetics, medicine; bioremediation, human cell clones; Genetically Modified (GM) crops, GM food, and animal cloning. The first successful cloning was conducted by a group of scientists at Roslin Institute, Scotland, in 1995, using two sheep, Megan, and Morag.

The following year, the same group of scientists used adult stem cells to clone Dolly. Presently, scientists are not only cloning various species of animals, but these advanced scientific techniques are also used for genetic modification (GM) purposes, such that in the production of transgenic animals ( Hasim et al., 2020 ). This process entails introducing a foreign gene into an animal’s genome to deliver desirable and economically significant characteristics in an animal. For instance, an experiment attended to produce a sheep that expresses a human gene resulted in the Factor IX protein in its milk, which can then be used to treat blood clots in humans with haemophilia ( Ibtisham et al., 2017 ). Similarly, transgenic sheep are also made to produce human alpha-1-antitrypsin, which can treat emphysema diseases ( McCreath, 2000 ). Other goals of cloning include the production of genetically modified animal organs to support human compatibility. Following the recognition of the numerous advantages of cloning and its capacity to serve various objectives, the agricultural sector has incorporated animal cloning into its practices to promote economic and environmental factors. Some typical transgenic methodology applications in agriculture comprise advanced milk production quality, improved disease resistance, and enhanced carcass composition to reduce environmental impacts ( Isa, 2013 ). In attempts to reduce the environmental impacts, scientists are also producing featherless chickens to reduce overall farming costs and pigs with a lower amount of phosphorous in their faeces ( Thomas, 2003 ). Scientists at Texas A&M University have also cloned a cow resistant to brucellosis ( Phillips, 2002 ).

US Food and Drug Administration (FDA) conducted extensive assessments to evaluate cloned animal food products’ safety. It was shown that there is no difference in the composition of food products produced from animal clones and their offspring in terms of food safety relative to conventionally bred animals ( FDA, 2008 ). Besides, a literature survey that analysed the composition, quality parameters, genotoxicity, and allergic reactions observed no differences in these parameters between meat or milk derived from cloned animals and their progeny from meat and milk of its nonclone counterparts ( Hur, 2017 ). No further evidence was shown that meat and milk from cloned animals pose a food safety risk. Thus, these findings were following the evaluations from the FDA.

The development of cloning technology has triggered severe concerns and garnered countless controversies surrounding ethical and religious perspectives ( Isa, 2013 ). This study concentrated its arguments based on Islamic rulings, as it is the official religion of Malaysia. Allah SWT said: And We have not sent you, [O Muhammad], except as a mercy to the worlds (al-Anbiyaa’, 21:107). This verse symbolises that Islam was sent down as a mercy to humankind, supporting our understanding that the basis of Islamic rulings considers society’s interest ( Samsudin et al., 2015 , Hasim et al., 2016 ). Consequently, modern biotechnology’s commercialisation and utilisation are also essentially dependent on the public’s perception and approval of stated technology ( Amin et al., 2009 ). This study analysed modern biotechnology, primarily about foods from cloned animals, in the scientific and Islamic context.

2. Animal cloning

2.1. natural cloning.

There are several methods of animal reproduction, including asexual and sexual reproduction. Asexual reproduction coexists with hermaphroditism and bisexual internal and external sex ( Benagiano and Primiero, 2002 ). Animals may reproduce through asexual means by budding in jellyfish, coral reefs, tapeworms; fragmentation in worms; and parthenogenesis in fish, insects, frogs, and lizards. Most animals that reproduce asexually do so through parthenogenesis, which is triggered during specific conditions. Parthenogenesis is a more effective form of breeding than sexual reproduction, as it enables faster exploitation of available resources ( Vajta and Gjerris, 2005 ). Nevertheless, mammalian asexual reproduction is not a naturally occurring phenomenon, despite the possibility of monozygotic twins (genetically identical) in mammals. Monozygotic twins are not considered clones as they are not the product of asexual breeding, and they differ from cloned animals, which only share the core DNA (different mitochondria) ( Vajta and Gjerris, 2005 ). Hence, the cloning of animals, primarily livestock animals, is a relatively new phenomenon.

2.2. Somatic Cell Nuclear Transfer (SCNT) technique

Over the last 20 years, Somatic Cell Nuclear Transfer (SCNT) has become indispensable in stem cell research with considerable potential in producing SCNT cloned animals. This technique is widely used to produce cells and tissues that are immune-compatible to the somatic cell donor ( Matoba and Zhang, 2018 ). SCNT appeared as brand-new biotechnology through which the possibilities derived from the advancements in molecular genetics and genome analysis in animal breeding. So far, more than 20 mammalian species have been cloned since the success of the first cloned mammal, Dolly the Sheep ( Matoba and Zhang, 2018 ).

The SCNT technique entails three essential steps: oocyte enucleation, implanting donor cells (or nucleus); and the reconstructed embryo ( Vajta and Gjerris, 2005 , Niemann, 2016 ). The cloned embryos are cultured in-vitro for some time, and once at their optimal level, the embryos are then transferred into the ‘parent’ animal ( Isa, 2013 ). In cloning, the nuclear genome (DNA) of a cell is replaced with another. The process is commenced by removing the maternal DNA from the mature oocyte, which is then replaced by the donor cell DNA ( UNESCO, 2005 ). Somatic cells may be derived from the animal, from cells grown through culture media or frozen tissues ( Committee on Science, Engineering and public policy, national academy of sciences, national academy of engineering, Institute of Medicine, 2002 ). A combination of chemicals is then introduced to prompt fertilisation, which results in the blastocyst stage. The derived embryo is then transferred or implanted into the uterus of an animal, followed by the natural process of pregnancy and birth ( Isa, 2013 ).

Animals bred through natural sexual reproduction contrasts from cloned animals that are a by-product of a combination of two random genes ( UNESCO, 2005 ). There are two possibilities. Firstly, if the ovum used is from the nucleus donor’s mother, or the nucleus of the donor itself ( Ayala, 2015 ). The resulting clone will hold the same genes (from the same nucleus and mitochondria) of the mother. The second possibility is if the ovum and nucleus used are from two distinct animals, or animals with different mothers. The resulting clone will then have a different gene as the genes are from differing mitochondria ( Ibtisham et al., 2016 ).

The success rate of animal cloning carried out by scientists, is still mostly inconsistent, with results profoundly dependent on the species and the type of cells used in the process. SCNT performance is relatively low, with success rates of 0.3–1.7 per cent per reconstructed oocyte and 3.4–13 per cent per transferred SCNT embryo ( Burgstaller and Brem, 2017 ). While complete nuclear transfer has been successfully cloned numerous mammals and has improved cloning performance, the proportion of cloned embryos that grow to full term remains poor, limiting the application of nuclear transfer technology ( Czernik et al., 2019 ). Furthermore, the cloned foetus miscarriage commonly occurs with a significant increase in the risk of foetus abnormality or mortality. Even after birth, developmental abnormalities remain in cloned mammals ( Loi et al., 2016 ).

The abnormalities and malformations resulted in the poor performance of SCNT that can be termed as the Large Offspring Syndrome (LOS), where the most commonly noted anomaly include the mismatch of size (cloned animals are too large for normal birth), as well as placental growth abnormalities ( Harris, 1997 , Ibtisham et al., 2017 ). LOS is now generally used in the discussion of other malformation and diseases. Besides the already existing complications, the unexpected mitochondrial dysfunctions in cloned embryos complicate the cloning process. Thus, it reduces the success rate ( Czernik et al., 2017 ). Several initiatives have, therefore, been implemented to boost the effectiveness of SCNT. These improvements include the technological aspects and the targeted alteration of the donor nucleus before or after the embryo’s development ( Czernik et al., 2016 ).

3. The application of animal cloning in agriculture

The recent advances in cloning efficiency have enabled diverse applications of SCNT technology. The advancement of SCNT in agriculture enhances the propagation of breeding farm animals and preserve the genetic resources of commercially important species, including cows and sheep ( Gomez et al., 2009 , Keefer, 2015 ). The weight of SCNT in the agricultural sector is more significant than in biomedicine. While the scientific and technological challenges of SCNT in both sectors are similar, employment in agriculture is more productive due to environmental variability and economic factors, such as cost efficiency, unique to agriculture. Cloning can be used to produce animals with desirable traits to yield healthier milk and meat for human consumption ( Paterson et al., 2003 ). The study administered by Takahashi & Yoshihio (2004) compares a sample of meat from cloned embryos, somatic clones and naturally produced animals, indicated no significant biological differences amongst the sample.

Genetically modified clones are considered more desirable than its traditionally bred counterpart, as clones tend to possess improved qualities such as healthier milk, meat and disease-resistant properties, resulting in a flow-on effect to benefit the wider population ( Vajta and Gjerris, 2005 ). One of the limitations of cloning in agriculture is its inability to produce consistent breeding animals’ results with the aspired traits ( Isa, 2013 ). It can be explained by the absence of a consistent mitochondrial DNA, as mitochondrial DNA varies according to the donor eggs. Also, the primary explanation for the low cloning competence is assumed to be the inability to reprogram the donor genome ( Rodriguez-Osorio et al., 2012 ). Further studies to enhance animal cloning efficiency such as bovine are needed to optimise the SCNT stage with an augmented recognition of the reprogramming mechanism ( Akagi et al., 2014 ). Moreover, the implementation of this technology depends not only on the animal’s genetic merits but also on the public perception and widespread acceptance of said technology ( Vajta and Gjerris, 2005 ).

4. Risk of the animal product derived from a cloned animal

The safety and ethical concerns associated with the products derived from modern biotechnology, especially cloned animals, are still controversial subjects ( Hasim et al., 2020 ). Nevertheless, previous studies have shown that animal products’ chemical composition, including meat and milk, is similar between clone-derived and nonclone-derived animals ( Hur, 2017 ). Most animal studies published that consuming meat and milk from cloned animals did not cause health problems and did not produce toxic effects. Dietary meat and milk derived from cloned animals also caused no adverse health effects such as reproduction and allergic reactions in animal models. Therefore, cloned animal meat and milk are as safe as food from their noncloned counterparts and can be consumed as novel foods ( Hur, 2017 ).

5. Islamic perspective on animal cloning

With the evolution of animal cloning in agriculture, naturally, the discussion of consuming foods from cloned animals is prompted. As a by-product of modern biotechnology processes, food is a comparatively brand-new concept, which requires new rulings that are more in line with the current developments. Before determining whether it is permissible or forbidden – haram or halal – to be consumed, this food category needs to be critically examined. Through the guidance of al-Qur’an and al-Sunnah (traditions of Prophet Muhammad PBUH), Muslims have relied on clear guidelines to determine the legality of matters. The general parameter on which the permissibility of a matter is based on is that unless something has been proven haram, or possessing haram features, Islam perceives the matter to be halal and permissible. The fiqh (Islamic jurisprudence) states:

الأصل في الأشياء الإباحة حتى يدل الدليل علي التحريم

“The original (basic) law for everything is permitted, unless there is an indication that shows the forbidden state of it.” ( al-Suyuti, 2001 , Nujaym, 1985 ).

Generally, Islam bans matters that are detrimental to one’s self. Thus, according to Islamic jurisprudence, the rulings for food as a by-product of modern biotechnology processes such as animal cloning should be determined based on the effects of its consumption by humans and whether it breaches any shariah principles. Following this belief, Muhammad Sulaiman al-Ashqar (2006) established the importance of Islamic organisations in examining the effects of food and medicinal products to provide a clearer understanding that can determine the permissibility of said food. Besides, the muftis are also accountable for researching animal-based food products, especially those produced through modern cloning methods ( Arifin, 2019 ). Hence, the Islamic authority needs to engage in extensive reviews regarding modern biotechnological processes, including animal cloning, to decide its permissibility in an Islamic context.

5.1. The determination of the permissibility of food derived from cloned animals

The permissibility of food’s analysis in this study is limited to the subject of food as a by-product of the modern biotechnological process of animal cloning. It is because cloning is generally performed as a means of breeding, for human consumption. It is vital to ensure that all new animal-based products produced through modern biotechnology must comply with the Quran and Sunnah requirements. ( Kashim et al., 2020 ). The views of the fuqaha’ regarding the permissibility of food derived from animals suggest that six principles could be used as a guideline in concluding the rulings of cloned animal-derived food. The principles are:

• a) Principle one: Halal and haram animals

A modern food product produced from halal animals is deemed halal ( Husni et al., 2015 , Kashim et al., 2018a , Kashim et al., 2018b ). Following this principle, any food produced through biotechnological processes to cater to the modern Islamic community should first assess the permissibility (halal or haram) of the types of animals that form the basis or foundation of the developed food product ( Husni et al., 2012 ). It is fundamental to ensure that the benefits of food products produced through modern biotechnology application could be preserved ( Al-Bakri, 2019 ).

• b) Principle two: Islamic process of animal slaughtering

There are essential conflicts of opinion between the Fuqaha’ in regards to the concept of al-dhakah . It happens due to the distinct understanding of the dalil for the process of slaughtering found in the Quran, the Sunnah, or through the practices of the Sahabahs ( al-Tariqi, 1983 , Rahman et al., 2018a , Rahman et al., 2018b ). The process of slaughtering requires the rupture of three critical veins: halqum (trachea); mariy’ (oesophagus) ; and wadajay (jugular). Therefore, cloned animals should benefit the Islamic community in every aspect and be slaughtered according to these principles ( Kashim et al., 2017 ).

• c) Principle three: Not derived from a source of najis (impurity)

Cleanliness is one of Islam’s most critical aspects. Hence, it should also be considered in producing food derived from cloned animals ( Rahman et al., 2018a , Rahman et al., 2018b ). According to the Islamic Shariah, there are various sources of Najis (impurity), such as carcasses that were not slaughtered or animals that were cloned from an animal that is classified as Najis . For a cloned animal-derived food product to be defined as clean, and not a Najis , it should adhere strictly to the conditions as set out in the Islamic law, and not be contaminated with any sources of Najis , including flowing blood ( al-masfuh ) ( Kashim et al., 2015 ). According to Malaysian Standard (2019) , in MS1500:2019 document, najis is defined as matters that are impure according to Shariah law and fatwa ( Kashim et al., 2017 ).

Adherence to Islamic law should be emphasised as many studies attended on animal cloning that does not comply with Islamic law. For instance, transgenic paddy production requires cloning pig DNA in paddy plants that aim to develop paddy plants resistance to herbicides, which could increase rice production ( Kawahigashi et al., 2005 ). Additionally, there is also cloning of rat genes in potato trees for the same purpose ( Yamada et al., 2002 ). These animal cloning products are contrary to Islamic rules because there has been a mixing of haram and halal sources. On that basis, Malaysia’s mufti has banned such cloning to protect Muslims’ rights (Federal Territory Mufti, 2020).

Blood is often used in food processing. There are two contrasting forms of blood, the flowing and non-flowing blood. Both hold different laws. The four Madhab jurists have banned its use in all food products for the flowing blood, including GMOs. While non-flowing blood such as the liver, spleen, blood attached to animal flesh is halal eaten by Muslims (al-Nawawi t.th; al-Zuhayli 1998). Therefore, non-flowing blood in the processing of animal cloning products is considered halal following Islamic law (Federal Territory Mufti, 2020). Based on the fatwa, cloning-based food products from animal sources should be determined on a case-by-case basis. Consequently, in the legal issue of a clone-based product’s impurity, it needs to be decided based on the origin of the material taken.

• d) Principle four: Istihalah tammah (perfect substance change)

The concept of Istihalah closely relates to aspects of cleanliness and purity ( taharah ), especially in the discussion of the modern biotechnological process of animal cloning. The Prophet Muhammad PBUH characterised cleanliness as one of the sources of Iman (faithfulness) of a Muslim (Qazzafiy, 2008). Istihalah tammah in animal cloning derived food is essential due to its purpose to purify impurities from contaminated substances. This process removes impurities from the originating body after it has been transferred into the new body.

The process of Istihalah , to purify Najis -contaminated substances, can either take place naturally or through human intervention. Substances’ status that was previously deemed haram could be changed to become halal and, in turn, be optimally used in various industries. For instance, the consumption or use of wine in food is haram. Nevertheless, through the process of istihalah , the wine can be fermented and turned into vinegar, which is halal to be consumed and used in food. Vinegar is considered halal, in contrast to its haram original form (wine), as the characteristics associated with wine, such as its smell, taste, and colour can no longer be identified. In line with this concept, food derived from the modern biotechnological process of animal cloning may be categorised as halal, given that it undergoes the process of istihalah tammah ( Kashim et al., 2019 ).

• e) Principle five: Maslahah (public interest) and mafsadah (damage)

The Islamic jurisprudence ( fiqh ) scholars have defined the maslahah concept (public interest) as a method to confirm the permissibility of a matter based on serving the interest of the Muslim community – whether it is useful or poses harm ( al-Ghazali, M.M., 1992 , Abd and al-Salam, 2000 ). Al-Shatibi (1997) described maslahah as a process to ensure the continuity and livelihood of the human life, while other Islamic jurisprudence scholars defined maslahah as a necessity allowed by the shariah, to preserve one’s faith, soul, intellect, family and wealth ( Kashim et al., 2019 ).

The Ulama’ (Islamic scholars) agreed that in assessing the maslahah (public interest), and ultimately the permissibility of a matter, the interests to be served must satisfy the requirements of the Shariah (Islamic law). The maslahah (public interest) concept as a basis of law, must consider the five most influential factors that need to be preserved: religion, life, intellect, family and wealth ( al-Shatibi, 1997 ).

Mafsadah (damage), on the other hand, is a notion that is contrary to maslahah and is defined as something that causes harm in society, and which has been denied by Islamic law, due to its unfavourable impressions on religion, life, intellect, family and wealth ( Ibn Ashur, 2007 ).

Islam places high importance on the maslahah (public interest) of its followers in all aspects of life, including the effects of food produced through the modern biotechnological process of animal cloning ( Isa, 2013 ). The guideline in determining the permissibility of a matter in the context of Islamic law is commonly following the Islamic jurisprudence objectives of benefiting humankind and preventing harm from them. In the context of food, Allah has ordained upon Muslims to consume healthy, beneficial food while avoiding the contaminated and unhealthy food ( Kashim et al., 2020 ). However, in studying the maslahah of a matter, the benefits or the public interest should always adhere to the conditions set out in Shariah (Islamic law), to prevent the abuse of the concept of maslahah .

Based on researchers’ discussion on the maslahat and mafsadah, contemporary scholars have approved all types of animal cloning processes that lead to maslahat, such as medicines’ production to preserve human life (Maqasid al-Syariah). Islam also supports cloning in the agricultural sector if it can positively impact a country’s economy and as long as it does not abuse the transgenic animals (Federal Territory Mufti, 2020).

Nevertheless, contemporary scholars have banned all types of cloning processes that cause mafsadah, which induce harm to humans and animals ( Arifin, 2019 ). Islamic scholars in human cloning domain have declared an absolute ban. It is because human cloning does not meet the needs of maslahat but instead leads to greater mafsadah. For instance, ideas and studies on human cloning have insulted human glory created only by Almighty God. It worsens when cloning against humans will endanger lives and found various criminal offences in the future. Thus, prevention is better than cure ( Kashim et al., 2020 ).

f) Principle six: Darurat (exigency) of animal cloning

Darurat (exigency) refers to a situation that necessitates immediate action, and at which people often act irrationally and perform prohibited acts to protect their religion, soul, mind, family and wealth (al-Suyuti, n.d.). The fuqaha (scholars) have agreed that any prohibited acts done to preserve the abovementioned five maqasid , during an exigent period is exempted and is considered halal (permissible) ( al-Ramli, 1987 , Ibn et al., 1979 ). Nonetheless, to prevent the random and liberal use of this exclusion, the determination of a Darurat (exigent) situation should be in accordance to the conditions set out in Shariah (Islamic law) (Muhammad Adham, 2001; Rahman et al., 2019 ).

6. Conclusion

Animal cloning is a comparably new phenomenon which has amassed critical attention and research by scientists. The evolution of this technology renders imperative benefits, particularly in the biomedicine and agriculture sectors. In biomedicine, the advancement of SCNT could develop animal models to study the pathogenesis of human diseases and established genetically engineered xenograft organs for patient transplantation. Still, as much as numerous benefits it offers, animal cloning is exposed to some risks, including deformation and abnormalities related to the Large Offspring Syndrome (LOS). In addition to scientific concerns, animal cloning’s biotechnological process also exhibits some ethical issues such as ‘playing God’ and the technology’s abuse to clone other humans. From an Islamic viewpoint, the rulings of animal cloning’s permissibility could vary according to current circumstances and fatwas . The permissibility of animal cloning in Islam’s context depends essentially on its impacts on the Muslim community’s interests ( maslahah ) and whether there is an exigent need ( Darurat ) for said process.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We thank the research project grant code FRGS/1/2019/SSI03/UKM/02/1 and FRGS/1/2017/SSI12/UKM/01/1 by Ministry of Higher Education, Malaysia for the supports.

Peer review under responsibility of King Saud University.

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Introduction

Openvoice v1.

As we detailed in our paper and website , the advantages of OpenVoice are three-fold:

1. Accurate Tone Color Cloning. OpenVoice can accurately clone the reference tone color and generate speech in multiple languages and accents.

2. Flexible Voice Style Control. OpenVoice enables granular control over voice styles, such as emotion and accent, as well as other style parameters including rhythm, pauses, and intonation.

3. Zero-shot Cross-lingual Voice Cloning. Neither of the language of the generated speech nor the language of the reference speech needs to be presented in the massive-speaker multi-lingual training dataset.

OpenVoice V2

In April 2024, we released OpenVoice V2, which includes all features in V1 and has:

1. Better Audio Quality. OpenVoice V2 adopts a different training strategy that delivers better audio quality.

2. Native Multi-lingual Support. English, Spanish, French, Chinese, Japanese and Korean are natively supported in OpenVoice V2.

3. Free Commercial Use. Starting from April 2024, both V2 and V1 are released under MIT License. Free for commercial use.

OpenVoice has been powering the instant voice cloning capability of myshell.ai since May 2023. Until Nov 2023, the voice cloning model has been used tens of millions of times by users worldwide, and witnessed the explosive user growth on the platform.

Main Contributors

• Zengyi Qin at MIT and MyShell
• Wenliang Zhao at Tsinghua University
• Xumin Yu at Tsinghua University
• Ethan Sun at MyShell

Please see usage for detailed instructions.

Common Issues

Please see QA for common questions and answers. We will regularly update the question and answer list.

Join Our Community

Join our Discord community and select the Developer role upon joining to gain exclusive access to our developer-only channel! Don't miss out on valuable discussions and collaboration opportunities.

OpenVoice V1 and V2 are MIT Licensed. Free for both commercial and research use.

Acknowledgements

This implementation is based on several excellent projects, TTS , VITS , and VITS2 . Thanks for their awesome work!

Contributors 13

• Python 86.6%
• Jupyter Notebook 13.4%