Nature Genetics
36, 775 - 780 (2004)
Published online: 6 June 2004; | doi:10.1038/ng1373
Sequential targeting of the genes encoding immunoglobulin- and prion protein in cattleYoshimi Kuroiwa1, 2, Poothappillai Kasinathan3, Hiroaki Matsushita3, Janaki Sathiyaselan3, Eddie J Sullivan3, Makoto Kakitani2, Kazuma Tomizuka2, Isao Ishida2
& James M Robl31
Gemini Science, 3030 Bunker Hill Street #226, San Diego, California 92109, USA. 2
Pharmaceutical Division, Kirin Brewery, 26-1, Jingumae 6-chome, Shibuya-ku, Tokyo, Japan. 3
Hematech, 4401 South Technology Drive, Sioux Falls, South Dakota 57106, USA.
Correspondence should be addressed to Isao Ishida i-ishida@kirin.co.jp or James M Robl jrobl@hematech.comGene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin- (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.
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