Gene manipulation strategies
Genetic strategies for genome manipulation
A number of methods have been developed to introduce genetic modifications into animals of research. Of those methods, virally mediated genetic modification is the only one that has been successfully utilized in the zebra finch. Viral-mediated gene delivery uses the natural ability of viruses to introduce genetic material into cells. The virus can be engineered to carry genetic material that can serve a number of different purposes. For instance, a gene encoding a fluorescent protein to image the morphology of neurons, a mutant human gene to create an animal model of a disease, an RNAi construct to reduce expression levels of an endogenous gene, or a channel to electrically activate or silence a specific neuronal cell population of interest. The introduction of this genetic material can be restricted to a population of cells by locally injecting the virus into particular brain regions, or more broadly, by targeting germ cells that will give rise to progeny carrying the transgene in every cell of the body (i.e. germline transgenics).
The virus used for producing germline transgenics is a lentivirus, a type of retrovirus capable of infecting both dividing and non-dividing cells. In addition, unlike other retroviruses, it escapes transcriptional silencing by host cell defense mechanisms. The virus can carry up to 10 kb of genetic material, so this must be taken into account when considering introducing genes that are quite large. The method used to generate transgenic zebra finches involves injecting the virus into the early embryo to target precursors to the germ cells. Ideally, the earlier the injection the better. However access to the embryo for the first 24 hrs after fertilization (which occurs intra-utero) is highly limited. As a result, injections are made after the egg is laid when the embryo is made up of tens of thousands of cells. At this time, the embryo is essentially a disc of cells with a diameter of about 0.5 mm and 1-2 cell layers thick. It is estimated that precursors to the germ cells number between 50-100 and are localized to the central portion of the disc. Thus, viral injections are targeted to this region.
Key issues/observations/comments regarding this procedure:
1) For unknown reasons, the extent of cellular infection in zebra finch embryos is substantially less than what has been observed in chicken and quail. This makes the infection of germline precursor cells less efficient. We think that one problem associated with this is viral access to cells. In quail and chickens, a single penetration/injection of the virus is sufficient to infect many cells. However, in the zebra finch multiple penetrations/injections are required to label a sufficient number of cells, suggesting a barrier of some sort restricting the virus's diffusion and/or ability to directly contact cells. Of course, there are many steps involved in the infection process and these must also be considered when attempting to explain the differences observed.
2) In addition to requiring multiple penetrations/injections, it is also necessary that the viral titer be quite high. Viral titer is determined using HEK 293T cells and I would not use anything less than 3x106TU/ul. On the other hand, you should determine this empirically by injecting embryos, incubating them for about a week and then evaluating the extent of the infection. Importantly, we have found that different batches of high titer virus can give significantly different levels of infection. Thus, making several batches and comparing them is also recommended.
3) Finally, and unfortunately, this method is relatively inefficient for generating germline transgenic zebra finches. Given the importance of this technology to the field, it is imperative that improvements and/or alternative methods be developed. For those interested, it would be helpful to discuss this issue to see how we might overcome this limitation.
4) If you have any questions / suggestions please offer them. This way we can add and/or modify this section with information that we have not considered.
5) For a more indepth look at techniques for gene manipulation we have provided some references below.
Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Science. 2002 Feb 1;295(5556):868-72. Epub 2002 Jan 10.
**This reference describes the generation of germline transgenic animals using lentiviral vectors.
Scott BB, Lois C. Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16443-7.
**This reference describes the generation of transgenic poultry (quails) with lentiviral vectors
Agate RJ, Scott BB, Haripal B, Lois C, Nottebohm F. Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17963-7.
**This reference describes the generation of transgenic songbirds (zebra finches) with lentiviral vectors.
Scott BB, Velho TA, Sim S, Lois C. ILAR J. 2010;51(4):353-61.
**This reference discusses the potential uses of transgenic manipulatiuon in birds.
Scott BB, Lois C. Nat Protoc. 2006;1(3):1406-11.
**This reference provides a detail step-by-step protocol describing the strategy to generate transgenic birds. The protocol describes the procedures used to generate transgenic poultry, but it can be easily adapted for songbirds.
Lentiviral transgenics introduce genes randomly in the genome. Stem cells allow targeted genome integration, with recombinant knock out of genes of interest. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. We have been working for the past two years (2009-2011) to generate iPSC of songbirds. We have been able to generate iPSC of zebra finches and chicken, and have so far gotten the chicken cells to form chimeric embryos. Ricardo Antonio Rosselló, Chun-Chun Chen, Rui Dai, Jason T Howard, Ute Hochgeschwender, Erich D. Jarvis.