Discovery Talk Questions and Answers: The Birth of Gene Targeting

Mario R Capecchi
Assessments created by Dr. Yi Liu

Questions

At the beginning of his talk, Dr. Capecchi defines gene targeting. Which of the following statements are true, according to his explanation? (Select all that apply).
1. Gene targeting is a method to change any gene in any conceivable manner in an organism.
2. Gene targeting allows us to selectively inactivate a particular gene in the mouse
3. Gene targeting allows one to deduce the function of a gene by looking at the phenotype after the gene’s deletion.
4. None of the above
Dr. Capecchi told the story about the pioneer experiment of gene targeting by Wigler and Axel both in his talk and in the paper. This experiment successfully introduced DNA into mammalian cells through DNA calcium phosphate co-precipitation. What gene did they introduce into the mammalian cell in that experiment?
1. Neor (Neomycin Resistance gene)
2. Hprt1 (Hypoxanthine Phosphoribosyltransferase)
3. HSV-tk (Herpes Virus Thymidine Kinase)
4. None of the above
Dr. Capecchi demonstrated that the increased the transforming capacity of by viral enhancer sequences resulted from… (Select all that apply)
1. Independent replication of the plasmid inside the cell
2. Increased integration into the genome
3. Increased expression of the integrated sequence in the host genome
4. None of the above
In his talk and the paper, Dr. Capecchi refers to concatemer.
Define “concatemer” in this context.
Dr. Capecchi proposed two possibilities for generating large concatemers and proved one to be correct. Which of the following statement describes the correct mechanism?
1. Random ligation
2. Rolling cycle replication
3. Homologous recombination
4. None of the above
Why was the discovery of a concatemer so important to Dr. Capecchi and his colleagues?
Dr. Capecchi mentioned in his talk that he submitted a grant proposal to NIH around gene targeting in the early 1908s, but got rejected. Explain the reason of this rejection, and how Dr. Capecchi had planned on addressing this issue.
In his talk.
Dr. Capecchi describes an approach to select cells that are correctly targeted: (9:07) “An example would be we have a defective gene copy already in the genome, and we’ll add a copy of that same gene with a different defect. Either one by itself would not be functional but together, by homologous recombination, they could recombine in such a way that now they would give you a functional copy because there are different mutations on those separate genes. And so that allows…and if that gene is required for the cell to survive, then you have a very strong selection that may work…be able to pick up events, one in a million or so.” Identify the figure in the paper that illustrates this approach, define the target gene and describe the selection process.
Dr. Capecchi referred to EC cells, ES cells and EK cells in his talk.
1. How are they referred to in his paper? Please list the full names of these cells.
2. Discuss the reason why Dr. Capecchi switched from EC cells to ES cells for gene targeting.
List 4 important milestones in the development of gene targeting.

Answers

a, b and c
Video (0:22): “First of all, what is gene targeting? It’s a method, essentially, of being able to change any gene in any conceivable manner in an organism. And our particular organism is the mouse. And so what we want to do, the mouse has many genes, 30,000 genes. And this allows one to selectively inactivate a particular gene and for example, if a little finger disappears then we know when the program for making a little finger is. And then that way, be able to deduce, essentially, what each gene is doing by what outcome to the mouse is…when we modify a particular gene.”
c
p. 507: “Wigler and Axel had just demonstrated that mammalian cells deficient in thymidine kinase (tk–) could be transformed into tk+ cells by exposing these cells to a DNA calcium phosphate co-precipitate containing the herpes virus thymidine kinase (HSV-tk; also known as HHV4gp124) gene2.”

Video (1:12): “Richard Axel and Wigler had shown, essentially, if you make a precipitate of DNA and put them on top of cells, the cells eat the DNA and then a certain amount of it would then go into the genome and be functional. For example, if a cell is thymidine kinase minus, this is an enzyme that’s required for thymidine uptake. So if that gene isn’t there, they can supply it exogenously. And they add…make a precipitate, give it to the cells, and about 1 in a million cells actually, then, acquires this gene in functional form and becomes thymidine kinase positive.”
b and c
These sequences either increased 1) integration into the genome or 2) probability of expression of the gene in the host genome. However, they did not result from an independent replication of the plasmid.
p. 508: I showed that the enhancement did not result from independent replication of the injected HSV-tk DNA as an extra-chromosomal plasmid, but that the efficiency-enhancing sequences were either increasing the frequency of exogenous DNA integration into the host genome, or increasing the probability that the HSV-tk gene, once integrated into the host genome, was expressed in the recipient cells3,9.
When multiple copies of the plasmid are introduced into nuclei of mammalian cells, although they integrate randomly into one or a few chromosomal sites, they line up and form highly ordered head-to-tail structure. This DNA structure is called concatemer.Wikipedia: A concatemer is a long continuous DNA molecule that contains multiple copies of the same DNA sequences linked in series.
p. 508, Figure1 Legend: “When multiple copies of a DNA sequence (arrows) are introduced into mammalian cells (a), they are efficiently integrated into one or a very few random site(s) within the host genome as a concatemer.”

p. 508, Paragraph 2: “When many copies of the HSV-tk plasmid were injected into cells, although integrated randomly into one or two chromosomal sites, they were present within those sites as a highly ordered head-to-tail concatemer.”

Video (2:34) : “So we repeated those experiments and what we noted is if we put in multiple copies of the same DNA, what we found is that, again, that…all of that DNA went randomly into the genome but something very unexpected was seen. DNA has a direction; you read it from say left to right. And so, what we found is that all the DNA molecules were lined up next to each other in what we call a concatemer, a head to tail concatemer, they’re all in the same direction.”
C
p. 508: “We reasoned that such highly ordered concatemers could not be generated by a random ligation process, but could be generated either by replication (for example, a rolling-circle-type mechanism) or by homologous recombination between the newly introduced plasmid molecules. We proved that they were generated by homologous recombination11.”

Video (2:54) “And so, what we found is that all the DNA molecules were lined up next to each other in what we call a concatemer, a head to tail concatemer, they’re all in the same direction. Now, randomly, that’s impossible, because we would put in a thousand copies and a thousand copies would all be head to tail, head to tail, head to tail. So there were only two possibilities for how this could happen. One is that that the…one would act like a template and then like a sausage machine and then turn out more and more copies and it would all come out as one large concatemer, again head to tail. The other is by a process called homologous recombination. Where in essence, two molecules which have the same sequence can be split and be put together again and then again would have a head to tail concatemer by homologous recombination.”
The discovery of the concatemers allowed Dr. Capecchi and his team to demonstrate that the homologous recombination mechanism was involved in gene targeting. This would end up being essential in their future gene targeting experiments.p. 508: “This was the first demonstration that mammalian cells could mediate homologous recombination between newly added exogenous DNA molecules. This conclusion was significant because it showed that mammalian somatic cells, in this case mouse fibroblasts, contained an efficient enzymatic machinery for mediating homologous recombination. (…) It was immediately clear to me that if we could harness this machinery to carry out homologous recombination between a newly introduced DNA molecule of our choice and the cognate sequence in the recipient cell, we could mutate or modify almost any gene in mammalian cells in any desired manner.”
Dr. Capecchi believed that it would be possible to do gene targeting by leveraging the homologous recombination machinery in mammalian cells. His proposal was rejected because NIH argued that the chance of targeting the correct site in a 3 billion base pairs genome was too small. However, Dr. Capecchi explains that he was planning on selecting for cells that undergo correct recombination. Instead of giving up, Dr. Capecchi decided to pursue this line of research on his other funding and was successful.Video (8:27) “In 1984, we already presented data to say, “Well, now we want to do gene targeting(…) in cells.” We submitted a grant to the NIH. The NIH found that project not possible. They said, “The probability, essentially, of your piece of DNA ever being able to find that same sequence in 3 billion base pairs is impossible. I mean, the frequency would be much to low and therefore it’d never function.” And we realized that the frequency was going to be low and so what we were thinking about is simply developing as a part of a selection. An example would be: we have a defective gene copy already in the genome, and we’ll add a copy of that same gene with a different defect. Either one by itself would not be functional but together, by homologous recombination, they could recombine in such a way that now they would give you a functional copy because there are different mutations on those separate genes. And (…) if that gene is required for the cell to survive, then you have a very strong selection that may work…be able to pick up events, one in a million or so. And so that’s the way we were approaching it. But still, they were skeptical, they gave us money actually for other projects and what we did was to utilize that money to continue our effort in gene targeting. And fortunately, four years later, we had information that it actually was working. We sent the grant back to the same granting agency and they sent back a pink slip that said : “we’re glad you didn’t follow our advice.” So, that gives you an idea that, you know, if you have confidence in a particular idea go for it and see whether you can come through. It’s also risky in the sense that if four years later, we hadn’t had any results we would have been in deep trouble, and unable to obtain other grants simply because we had utilized those funds for something that didn’t work. Fortunately, four years later, we were successful and the project continued.”
p. 508: “In 1980, I submitted a grant proposal to the US National Institutes of Health (NIH) to test the feasibility of gene targeting in mammalian cells, and these experiments were rejected on the grounds that there was only a remote probability that the newly introduced DNA would ever find its matching sequence within a host cell genome. Despite the rejection, I decided to continue this line of experimentation.”
Figure2 illustrates this approach.The target gene is a neomycin resistance gene.
First, the defective neomycin resistance gene that contains a small deletion at the 3’ coding sequence was randomly integrated in the genome. Then, another defective neomycin resistance gene, which has a different mutation site (at the 5’ end of coding sequence) was introduced to the recipient cell. When a homologous recombination happens, the recipient cell chromosome can generate a functional copy of the neomycin resistance gene, making the cell resistant to the drug G418. Cells that didn’t undergo targeted homologous recombination would die.

p. 508, “The first step of this scheme required the generation of mammalian cell lines with random insertions of a defective neomycin resistance (neor) gene containing either a deletion or point mutation (FIG. 2). In the second step, target-vector DNA, carry- ing defective neor genes with mutations that differed from those present at the target site, was introduced into the recipient cell lines. Homologous recombination between neor sequences in the targeting vector and the recipient cell chromosome could generate a functional neor gene from the two defective genes, producing cells that are resistant to the drug G418, which is lethal to cells that lack a functional neor gene13.”
a. EC cells are embryonal carcinoma cells.
ES cells are embryonic stem cells. ES cells were also called EK cells in the 1980’s.
b. The ultimate goal for his gene targeting is to generate mice with gene modifications. Both EC cells and ES cells can contribute to somatic cell production, but EC cells cannot contribute to the germ line production, thus limiting the possibility of generating offspring with the same genetic modification. ES cells are better for gene targeting because they can be introduced into a pre-implantation embryo to mature in a foster mother mouse.

Video:(10:52) “And for this, at the time, the most attractive cells were called EC cells, embryonal carcinoma cells. And those are…it’s a tumor essentially, that’s made up of many multiple cell types and but, within them are stem cells, stem like cells in the sense that they could contribute to the formation of multiple different tissues. And so I was going from meeting to meeting looking at how the progress was being made with EC cells and it was sort of disappointing in a sense that it was working to contribute to tissues of the body, what we call somatic cells, but it wasn’t contributing to the germ line. And for us we wanted to go into the germ line, because then, if we ever made a modification, we could then generate as many mice as we want with that modification simply by breeding. But those cells didn’t exist. And then, fortunately, at around 1980…late 1984 I heard rumors that Martin Evans in Cambridge, England had actually started developing cells that may work. At that time he called them EK cells and those cells, what he did is to isolate, rather than isolating these cells from a tumor he isolated very similar cells from an embryo. And simply used EC cells as the driving force to say, “What kind of cells I want.” But now, instead of deriving it from a tumor he was isolating them from the embryo themselves and those cells looked like they may be capable of contributing to the germ line and therefore would be a suitable substrate for us to do gene targeting with.”

p. 509: “Instead, it seemed that our best option would be to carry out gene targeting in cultured embryonic stem (ES) cells, from which the relatively rare targeted recombinant would be selected and purified. When subsequently introduced into a pre- implantation embryo and allowed to mature in a foster mother, these purified cells would contribute to the formation of all tissues of the mouse, including its germline.”
p. 509: “Whereas ES cells were derived from early mouse embryos, EC cells were obtained from mouse tumours.”
1. To development of a method to efficiently transfer exogenous DNA into mammalian cells by microinjection
2. The discovery of homologous recombination machinery in mammalian cells
3. The development of a selection protocol for correctly targeted cells
4. The use of selected ES cells for gene targeting