Cloning and Transgenic Animals:
The Influence of Technical Confluence
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
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
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
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
When a researcher adds a donor gene to a population of cells,
there are at least three possibilities with each cell.
- The donor gene fails to insert.
- The donor gene randomly inserts anywhere in the genome.
- 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?
- The donor gene is usually not easy to find when it inserts.
- 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.
- 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.
- 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.
- 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
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?
- 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.
- 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.
- 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
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
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