Cloning and Transgenic Animals:

The Influence of Technical Confluence

Commentary by Thomas M. Zinnen

In 1997 'Dolly' and 'Polly' have changed how we look at cloning and transgenesis in multi-celled animals. Dolly, of course, is the famous 'cloned sheep' announced early this year, and 'Polly' is a transgenic sheep from the same research program.

Before 'Dolly' and 'Polly,' cloning animals and making transgenic livestock were lines of research that proceeded independently since the mid 80ís.

An early step in cloning in livestock was to split a developing calf embryo and generate two genetically identical calves from the two embryonic halves.

The first step in transgenesis as practiced by bacteriologists was to add a new gene to a cell in culture, and observe the effect of that one added gene, also in culture.

The successful birth and development of 'Dolly' the sheep showed that researchers could start with cells taken from adult animals, not just with embryonic cells. After the original cells were collected from the adult sheep, these cells were grown and multiplied in cell culture before being used to generate an embryo.

Growing and multiplying in culture populations of these totipotent cellsóindividual cells capable of going through embryonic and fetal development to produce an adult animalóis a powerful tool. Rather than manipulating one cell at a time, animal scientists are starting to be able to manipulate cell populations like the way bacteriologists do: adding genes to a population of cells and selecting for the particular rare cells that have incorporated the desired combination of genes.

Once that particular rare cell is found, it can be grown from a single cell to a complete animal. This means scientists can hope to study gene effects not just on cell physiology in a test tube, but also on growth and development into tissues, organs and complete animals.

Comparing what could be done in terms of transgenic animals before Dolly and what can be done using the 'Dolly technology' helps in understanding the impact that the 'Dolly technology' will have in research with transgenic animals.

Don't Just Add a Gene, Adjust It and Exchange It

The next step in research with transgenic livestock is site-specific insertion of genes using homologous recombination to study the effect of exchanging one version of a gene for another. Homologous in this case means that you replace a gene with a different gene for the same kind of function, located in the same place on the organismís genome.

Using homologous recombination is powerful because a researcher can make an inactive version of a gene and exchange it for an active version, to ask the question: 'What happens if I turn a gene off?'

But homologous recombination is to random insertion as a needle is to a haystack. The researcher is challenged to find that particular cell in which the newly donated gene trades places with a target gene already on a chromosome. The researcher needs to distinguish that rare cell from the cells which have the more common random insertion of the newly donated gene anywhere in the genome.

Imagine the problems of homologous recombination have been solved, but 'Dolly Technology' is not available. Researchers could routinely use a donor gene to exchange or 'knock out' the corresponding gene in a sheep cell. If the cells grow as undifferentiated tissue in culture, researchers could study the effect of the target gene on cell physiology. But researchers wouldnít know what, if any, function the gene may have in development or in the physiology of the adult animal.

Even if a researcher can find in a culture dish the rare cell with the rare homologous insertion event, how can the researcher generate a whole animal from that one cell in order to find out the geneís function in development?

To address those questions, one needs a system to take a single cell and make a complete animal. This is cloning. This is the breakthrough represented by 'Dolly Technology'. The reality is that 'Dolly Technology' is here, and the next big breakthrough will be a system of homologous recombination in livestock animals.

The State of Cloning and Homologous Recombination: 1997

So far, the only animal in which scientists have a system for inserting a known donor gene into a known site using homologous recombination is the mouse. Putting in a new version of a gene and "knocking out" the existing version of the gene is the basis of knock-out mice.

You can look at the term knock-out in two ways. The more general is 'one is put into place as the other is knocked out of place.' The version of the gene you put in might work but in a different way than the existing version of the gene it replaces. More commonly the donated gene is inactive, and in this sense the addition of the donated gene not only 'knocks out' the working copy of the gene, it can also knock out the normal geneís function.

In livestock, researchers can insert a known gene but the insertion is random. With livestock, it has been possible to add a new gene to an embryonic stem cell and then grow an adult animal from the cell. But so far it has been impossible to insert a new, modified version of an existing gene, replacing a normal version in a cell, and then generate an adult from that cell. A system of homologous recombination will allow researchers to study the effect of 'knocking-out' existing genes to study the genesí functions.

This is why homologous recombination will be so valuable in research on the genetics of livestock animals. Compare that with transgenesis. Transgenesis merely requires the random insertion of a new gene. The new gene inserts anywhere. It does not exchange or knock out of place any other gene.

Homologous Recombination in Transgenic Animals: The Mouse Model.

The challenge of harnessing homologous recombination is to get not random insertion but rather a specific exchange, where the donor gene is exchanged for and displaces the homologous existing resident gene.

When a researcher adds a donor gene to a population of cells, there are at least three possibilities with each cell.

1. The donor gene fails to insert.
   
   
2. The donor gene randomly inserts anywhere in the genome.
   
   
3. The donor gene by homologous recombination specifically inserts at the site of its homologous gene and displaces it.
   
   

This involves a significant technical challenge for the biologist: How to select for cells that have exchanged genes and select against cells that have received the donor gene through random insertion?

1. The donor gene is usually not easy to find when it inserts.
   
   
2. 1. Consider attaching to the donor gene a second gene that is easy to find.
      
      If you can find those cells in which the two connected genes have inserted into chromosomes, you have found those cells that have the target donor gene. A common gene for resistance is neo. It confers resistance to an antibiotic called G418. Keep in mind that the genes could be inserted randomly anywhere or they could be inserted by homologous recombination at the target site.
      
      
   2. Consider inserting the selectable neo marker into the donor gene, thereby inactivating the donor gene and yet it keeps its ability to undergo homologous recombination.
      
      
3. Consider attaching a third gene. Attach the third gene next to the donor gene. The third gene is next to but outside the region of homology. Consider the third gene to be a suicide gene or a counter-selectable marker that works opposite the way of a selectable marker. A suicide gene makes a cell vulnerable to a chemical or antibiotic. The standard example is the TK gene, or thymidine kinase gene. Cells that have and express the TK gene are killed by the antibiotic Gancyclovir. Cells without TK gene are not harmed by the drug.
   
   
4. Hereís the discerning part. If the donor DNA inserts randomly, then all three genes will be inserted: the inactive donor gene and the two active marker genes. That means the cell will both resist the first antibiotic (G418) but will be killed by Gancyclovir.

On the other hand, if the donor DNA inserts by homologous recombination, then only the homologous stretch of DNA is inserted. The flanking TK gene is outside the region of homology and it is not inserted. Instead it is jettisoned. The neo gene inserted within the donor gene is inserted, because it is between the two regions of homologous DNA. That means that cells with homologous recombination resist both G418 because they have neo and Gancyclovir because the cells lack TK gene.

Side Bar: Scoreable vs Selectable Markers Scoreable markers are an example of genes that are easy to find. Examples include a gene that makes an enzyme that makes a color (such as GUS) or that makes light (such as lux genes for luciferase) or that makes a fluorescent protein (such as the gene for Green Fluorescent Protein). The two genesóthe 'gene of interest' and the 'selectable marker'óare attached ('in tandem'). So where the scoreable marker goes, itís very likely the gene of interest goes too. Another example of a gene that is easy to find is a gene for resistance to an antibiotic. This is called a selectable marker. These are even more powerful than scoreable markers. Cells that receive a copy of the gene for resistance to the antibiotic kanamycin can grow in test tubes containing kanamycin, while other cells without the gene will die. With a scoreable marker, you may be able to pick out the one fluorescent cell in a thousand other cells that donít fluoresce. But with a selectable marker, only the cells with the marker remain alive after treating with the antibiotic. Itís the difference between an orange vest to make a cell stand out in a crowd and a bullet-proof vest that lets a cell survive the attack of antibiotics. Now you have your gene of interest and you can follow it using the attached selectable marker.

This means if you start with a population of cells growing in cell culture, you can generate and select for and obtain cells that have, through homologous recombination, exchanged a donor gene for an existing gene.

Until today, this means you can study the effect of the donor gene, but only in cells grown in tissue culture. And only in the heterozygous condition. Since each cell usually has two copies of each gene, the donor gene is likely working in the presence of the second copy of the original gene, on the sister chromosome.

(Heterozygous in this discussion means that the gene of interest is different in each sister chromosome. Homozygous means it is the same on each sister chromozome.)

Cloning: From single cell to whole animal.

How can you go from having a bunch of single cells with single copies of the donor gene in the presence of another copy of the original homologous gene, to having complete animals that are homozygous for the donor gene?

1. You need to grow a complete animal from the single cell that has one copy of the donor gene. That animal is heterozygous for the gene you're studying.
   
   
2. You need to breed that animal with another heterozygous animal. This is basically the same as crossing two animals both heterozygous for the gene of interest.
   
   
3. You need to test the offspring. Each of the offspring or progeny of the cross has a 25 % chance of being homozygous for the donor gene of interest. Finding which offspring are homozygous is easiest if there is a DNA fingerprint test that discerns between the donor gene and the original gene. With such a test, each progeny can be assayed, and those found to be homozygous for the donor are studied further.

How can a researcher grow a complete animal from a single cell, one that is not a zygote?

Hereís how it is done with pre-Dolly technology. Letís say you take an embryo at the 32-cell stage, and disrupt it into 32 individual cells. If you grow it in the appropriate medium and conditions, the cells will divide in culture. You can grow hundreds of these cells. These are called 'embryonic stem cells.' One of their key traits is that they can develop into any type of tissue while in culture: muscle, nerve, heart, and so forth.

You can also add a gene or two to embryonic stem cells. You can select for those cells that have undergone homologous recombination. You can take several of these modified embryonic stem cells and inject them into a second embryo, again at about the 32 or 64-cell state. You get an embryo with two genetically distinct types of cells: the prevalent original type, and the few that have the extra gene. As the mixed or 'chimaeric' embryo grows, all the cells derived from the modified embryonic stem cells have the extra gene; all the cells derived from the other cells do not have the extra gene.

If the cells that have the extra gene develop only into somatic tissues and organs and not into germline tissue, then the resulting adult chimaeric animal will give only offspring carrying unmodified genes. On the other hand, if the germline tissue derives from the modified cells, then the germline cells will have the extra gene and half of the gametes produced by the chimaeric animal will have the added gene. The drawback is you have to grow the animal to maturity to be able to test the sperm cells or ovae to see if they are carrying the gene of interest.

Mating a heterozygous male and a heterozygous female is expected to result in 1/4 of the progeny to be homozygous. The progeny can serve as founders for homozygous lines of animals that have knocked out a particular gene.

Sound complicated? It does to me.

How to simplify it?

Bypass the chimeric embryo using 'Dolly technology.'

Imagine if instead of starting with modified embryonic stem cells that have to be injected into a 32-cell embryo, you could instead take the modified embryonic stem cells and generate an adult animal from a single cell. That means all the cells of the embryo that develops from the original single cell will all have the copy of the gene.You would therefore bypass the chimaeric phase. Neither Dolly nore Polly would be chimeras.

You would still have to deal with a founder animal that is heterozygous. But this simplifies the system by reducing the need for one entire generation. Instead of chimaera to heterozygote to homozygote, you can go from heterozygote directly to homozygote. With mice, that is a savings of a few weeks. With livestock , that could mean a savings of 2 to 3 years.

This is the Anticipation of the 'Dolly' technology.

If you can grow an adult sheep from cells taken from an adult and grown in test tubes, then you can take a population approach: grow many cells, genetically manipulate them as a group, select for those cells that have the desired combination of traits. Take individual selected cells, and from them grow an entire adult. Breed that adult with another heterozygous adult, and you have a 1/4 chance of getting progeny that are homozygous for the inserted gene.

I anticipate that the news media will be dumbfounded when a researcher announces the development of a system of homologous recombination. I think the general public will have a hard time grasping the significance. With the cloning of 'Dolly' came the possibility of cloning adult humansóa long-standing topic of science fiction books and movies. But as far as I know, nobody has made any movies about homologous recombination. Itís important but sublime, even obtuse.

For geneticists, the sublime will be significant. From an applied point of view, modifying the genome of pigs by substituting the human version for the pig version of the major histocompatibility genes will 'humanize' pig organs, making them more likely candidates for transplanting to humans. From a fundamental point of view, modifying existing genes will speed our understanding of the roles of those genes in development from zygote to billy goat.

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