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Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

Nature Genetics volume 36, pages 775–780 (2004)Cite this article

Abstract

Gene 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-μ (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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Figure 1: Procedure for sequential gene targeting in bovine primary fibroblasts.
Figure 2: Sequential targeting of IGHM in primary bovine fibroblasts.
Figure 3: Removal of both neo and puro genes by Cre-loxP system.
Figure 4: Sequential targeting of PRNP in IGHM−/− fibroblasts.

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Acknowledgements

We thank J. Pommer, J. Koster, J. Molina and D. Faber for their assistance in embryo transfer, fetal recovery, calf delivery and sample collection and M. Nichols, J. Griffin, M. Bien, T. King, M. Ahlers, R. Paulson, S. Viet and C. Voss for their assistance in gene targeting and embryo cloning.

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Authors and Affiliations

  1. Gemini Science, 3030 Bunker Hill Street #226, San Diego, 92109, California, USA

    Yoshimi Kuroiwa

  2. Pharmaceutical Division, Kirin Brewery, 26-1, Jingumae 6-chome, Shibuya-ku, Tokyo, Japan

    Yoshimi Kuroiwa, Makoto Kakitani, Kazuma Tomizuka & Isao Ishida

  3. Hematech, 4401 South Technology Drive, Sioux Falls, 57106, South Dakota, USA

    Poothappillai Kasinathan, Hiroaki Matsushita, Janaki Sathiyaselan, Eddie J Sullivan & James M Robl

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  1. Yoshimi Kuroiwa
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  2. Poothappillai Kasinathan
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  4. Janaki Sathiyaselan
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  5. Eddie J Sullivan
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Correspondence to Isao Ishida or James M Robl.

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Competing interests

Y.K., M.K. and I.I. are employed by Kirin Brewery, which may gain or lose financially owing to publication of this paper. P.K., H.M., J.S., E.J.S. and J.M.R. are employed by Hematech, which may gain or lose financially owing to publication of this article. J.M.R. has a substantial financial interest in Hematech.

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Kuroiwa, Y., Kasinathan, P., Matsushita, H. et al. Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle. Nat Genet 36, 775–780 (2004). https://doi.org/10.1038/ng1373

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