I thought I had a post about the successful production of rat embryonic stem cells.
A recent paper in Science, reported the first knockout rat using engineered zinc-finger nucleases. I don't like this method because I don't trust these engineered ZFN: in this paper, the authors examined 20 predicted off-target sites and they were fine. But, what if the prediction is off? I like the precise way to engineer the genome - homology recombination.
Science. 2009 Jul 24;325(5939):433.
Knockout rats via embryo microinjection of zinc-finger nucleases.
Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R.
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
Both rES and riPS cell lines have been made. I think knockout Rat using these stem cells should come out soon.
Here are the two reports about generating embryonic stem cells from rat blastocysts.
Cell. 2008 Dec 26;135(7):1287-98.
Capture of authentic embryonic stem cells from rat blastocysts.
Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL, Smith A.
Institute for Stem Cell Research, University of Edinburgh, King's Buildings, Edinburgh EH93JQ, UK.
Embryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration.
Cell. 2008 Dec 26;135(7):1299-310.
Germline competent embryonic stem cells derived from rat blastocysts.
Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL.
Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.
Reports ealier about riPS
Cell Stem Cell. 2009 Jan 9;4(1):11-5.
Generation of induced pluripotent stem cell lines from adult rat cells.
Liao J, Cui C, Chen S, Ren J, Chen J, Gao Y, Li H, Jia N, Cheng L, Xiao H, Xiao L.
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Cell Bank, Stem Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Cell Stem Cell. 2009 Jan 9;4(1):16-9.
Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.
Li W, Wei W, Zhu S, Zhu J, Shi Y, Lin T, Hao E, Hayek A, Deng H, Ding S.
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Leave a Reply