Talk:CH391L/S12/CounterSelection

• Ben Slater 02:09, 25 February 2012 (EST): Does allelic exchange work in eukaryotes? It seems like it would be awesome for applications like gene therapy, if it allows chromosomal editing at any given site. Another question: how would one synthesize the homologous insert? Thanks!
  • David M. Truong 22:41, 26 February 2012 (EST): Joe and I have spent alot of time on this topic; he may or may not chime in. As far as I can tell, allelic exchange is merely homologous recombination, a technique developed for use in mammals by Mario Capecchi in the late 1980's, winner of the 2007 Nobel Prize [1]. However, the rate of natural homologous recombination in mammals is only 0.7 events per 10^6 [2]. Thus, selectable markers are often used to "find" the recombined gene, and even this can be fairly low, say 1% of selected cells. Therefore, natural homologous recombination for gene therapy can not work. Recently, the development of site-specific endonucleases such as Zinc-fingers [2] and TALEs [3] permit the increased efficiency of homologous recombination. By generating a double-strand break at the site of recombination these nucleases can increase the recombination rate to near 50% of all loci. In fact, zinc fingers against the CCR5 gene (an entry point of HIV) have already begun Phase II clinical trials. They have modified primary CD4 T-cells at rate of ~30% and infused them into AIDs patients. The effect they were hoping to see was that of the [Berlin Patient] who recieved CCR5(-) cells from a donor, and miraculously had these cells take over his blood stream and "cure" him of AIDs. Although promising, they have not achieved the miraculous effects seen in the Berlin Patient, only 1 patient has not seen recurrence of the virus. More generally, both Zinc-fingers and TALEs are difficult to modify towards new genes, requiring extensive protein-engineering, and sometimes have serious off-targeting issues that may lead to unwanted mutations in other genes (think cancer).
    • Yi Kou 23:13, 26 February 2012 (EST):nice example, there is one public database: Genetic Modification Clinical Research Information System GEMCRIS
  • Jeffrey E. Barrick 19:08, 26 February 2012 (EST):Here's an attempt at an answer. I'm no expert in this area. You can "transiently transfect" eukaryotic (mammalian) cells in culture by using viruses as vectors. This results in your gene being made, but isn't very stable -- it's like putting an unstable plasmid into a bacterium. You can also generate animals, like mice that have a gene knocked out. I'm not familiar with the exact methods, but it undoubtedly involves using selectable markers. In multicellular eukaryotes, a lot of the difficulty is also in getting the mutation in the germ line, or at least into cells that continue to divide during an organism's lifetime.
    • Yi Kou 20:43, 26 February 2012 (EST): I agree with Dr. Barrick's opinion. Clinically, there is no such application so far. Partially because of our comprehension of temporal and spatial arrangement of gene (& its regulation, expression) in multicellular organism is far from satisfying. Regarding temporal aspect, interruption of a gene can easily result in no observation of the expectation, simply because that the gene may be needed on one developmental stage and interruption of it leads to a termination of an observable maturity. For spatial factor, if a pivotal and universally needed gene has got somewhat modification, its phenotypic results can be seen anywhere in the organism, not leading to a reducible and conclusive manner. However, despite these, there have been developed several techniques, either improve "transfect" stability or make the independent variable more "independent" to be studied [4].
    • Adam Meyer 21:50, 26 February 2012 (EST):I am also no expert, but transient transfections are usually done with normal plasmids, not viruses. Viruses (commonly lentivirus-based) can be used for genome insertion in mammalian culture. This can yield stable lines after antibiotic selection.
    • Joe Hanson 12:45, 27 February 2012 (EST): Good points all around. To add to what Dave said, there are also ways to do allelic exchange in a manner that doesn't replace the original allele, but just introduces a corrected or modified version of it at an off-target site. Sort of like gene correction without recombination. It would be very similar to a dominant negative system in genetics. In terms of transient and non-transient expression methods, we could probably have a whole class about that. Lots of it is cell-type dependent, because only certain cell types are infected by certain viruses. But some viruses form transient expression vectors, others integrate the gene cassette for more stable expression and still others are in the middle with a bit of both. If anyone wants to discuss more in class, I'd be happy to, but it's of limited relevance to this topic.

References

<biblio>

1. paper pmid=20812916
2. Capecchi1987 pmid=2822260
3. Baltimore2003 pmid=12730593
4. Talens pmid=21738127