Abstract
We have used an insertion vector-based approach to target the Gi2α gene in AB-1 embryonic stem cells. 105 bp located 0.8–0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineeredNot I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained (‘hit’ step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy, 2-fluoro-β-d-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination (‘run’ step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified ‘hit and run’ strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated Gi2α allele is also demonstrated.
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Adair, G.M., Nairn, R.S., Wilson, J.H., Seidman, M.M., Brotherman, K.A., MacKinnon, C. and Scheerer, J.B. (1989) Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster ovary cells.Proc. Natl. Acad. Sci. USA 86, 4574–78.
Birnbaumer, L. (1990) G proteins in signal transduction.Ann. Rev. Pharmacol. Toxicol. 30, 675–705.
Birnbaumer, L. (1992) Receptor-to-effector signaling through G proteins: roles for βγ dimers as well as for α subunits.Cell 71, 1069–72.
Birnbaumer, L., Abramowitz, J. and Brown, A.M. (1990) Signal transduction by G proteins.Biochim. Biophys. Acta (Reviews in Biomembranes) 1031, 163–224.
Bradley, A. (1987) Production and analysis of chimaeric mice. In Robertson, E.J. ed.,Teratocarcinomas and Embryonic Stem Cells: a Practical Approach, pp. 113–51, Oxford: IRL Press Ltd.
Burch, R.M., Luini, A. and Axelrod, J. (1986) Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha1-adrenergic stimulation in FRTL-5 cells.Proc. Natl. Acad. Sci. USA 83, 7201–5.
Camerini-Otero, R.D. and Kucherlapati, R. (1990) Right on target.New Biologist 2, 337–41.
Corven, E.J. van, Groenink, A., Jalink, K., Eichholtz, T. and Molenaar, W.H. (1989) Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins.Cell 50, 45–54.
Deng, C. and Capecchi, M.R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.Mol. Cell. Biol. 12, 3365–71.
Endo, Y., Lee, M.A. and Kopf, G.S. (1987) Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction.Dev. Biol. 119, 210–6.
Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem. 132, 6–13.
Gilman, A.G. (1984) G proteins and dual control of adenylate cyclase.Cell 36, 577–9.
Gilman, A.G. (1987) G proteins: transducers of receptor-generated signals.Ann. Rev. Biochem. 56, 615–49.
Gurich, R.W., Codina, J. and DuBose Jr, T.D. (1991) A potential role for guanine nucleotide-binding protein (G protein) in the regulation of endosomal proton transport.J. Clin. Invest. 87 1547–52.
Hasty, P., Ramírez-Solis, R., Krumlauf, R., and Bradley, A. (1991a) Introduciton of a subtle mutation into the Hox-2.6 locus in embryonic stem cells.Nature 350, 243–6.
Hasty, P., Rivera-Pérez, J., Chang, C. and Bradley, A. (1991b) Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.Mol. Cell. Biol. 11, 4509–17.
Hasty, P., Rivera-Pérez, J. and Bradley, A. (1991c) The length of homology required for gene targeting in embryonic stem cells.Mol. Cell. Biol. 11, 5586–91.
Hasty, P., Rivera-Pérez, J. and Bradley, A. (1992) The role and fate of DNA ends for homologous recombination in embryonic stem cells.Mol. Cell. Biol. 12, 2464–74.
Imamura, K. and Kufe, D. (1988) CSF-1 induced Na+ influx into human monocytes involves activation of a pertussis toxin sensitive GTP-binding protein.J. Biol. Chem. 263, 14093–8.
Imamura, K., Sherman, M.L., Spriggs, D. and Kufe, D. (1988) Effect of tumor necrosis factor on GTP binding and GTPase activity in HL-60 and L929 cells.J. Biol. Chem. 263, 10247–53.
Itoh, H., Okajima, F. and Ui, M. (1984) Conversion of adrenergic mechanisms from an alpha- to a beta-type during primary culture of rat hepatocytes: accompanying decreases of the inhibitory guanine nucleotide regulatory component of adenylate cyclase identified as a substrate of islet-activating protein.J. Biol. Chem. 259, 15464–73.
Ito, H., Sugimoto, T., Kobayashi, I., Takahashi, K., Katada, T., Ui, M. and Kurachi, Y. (1991) On the mechanism of basal and agonist-induced activation of the G-protein gated muscarinic K+ channel in atrial myocytes of guinea pig heart.J. Gen. Physiol. 98, 517–33.
Jasin, M. and Berg, P. (1988) Homologous integration in mammalian cells without target gene selection.Genes Dev. 2, 1353–63.
Johnson, R.S., Sheng, M., Greenberg, M.E., Kolodner, R.D., Papaioannou, V.E. and Spiegelman, B.M. (1989) Targeting of nonexpressed genes in embryonic stem cells via homologous recombination.Science 245, 1234–6.
Kim, H.S. and Smithies, O. (1988) Recombinant fragment assay for gene targeting based on the polymerase chain reaction.Nucl. Acids Res. 16, 8887–903.
Kim, D., Lewis, D.L., Graziadei, L., Noer, E.J., Bar-Sagi, D. and Clapham, D.E. (1989) G protein βγ-subunits activate the cardiac muscarinic K+ channel via phospholipase A2 Nature 337, 557–60.
Kirsch, G., Codina, J., Birnbaumer, L., and Brown, A.M. (1990) Coupling of ATP-sensitive K+ channels to purinergic receptors by G-proteins in rat ventricular myocytes.Am. J. Physiol. 259, H820–6.
Kurachi, Y., Ito, H., Sugimoto, T., Shimizu, T., Miki, I. and Ui, M. (1989) Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel.Nature 337, 555–7.
Leyte, A., Barr, F.A., Kehlenbach, R.H. and Huttner, W.B. (1992) Multiple trimeric G-proteins on the trans-golgi network exert stimulatory and inhibitory effects on secretory vesicle formation.EMBO J. 11, 4795–804.
Mansour, S.L., Thomas, K.R. and Capecchi, M.R. (1988) Disruption of the proto-oncogeneint-2 in mouse embryoderived stem cells: a general strategy for targeting mutations to non-selectable genes.Nature 336, 348–52.
McMahon, A.P. and Bradley, A. (1990) The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain.Cell 62, 1073–85.
Mombaerts, P., Clarke, A.R., Hooper, M.L. and Tonegawa, S. (1991) Creation of a large genomic deletion at the T-cell antigen receptor β-subunit locus in mouse embryonic stem cells by gene targeting.Proc. Natl Acad. Sci. USA 88, 1084–87.
Mortensen, R.M., Zubiaur, M., Neer, E.J. and Seidman, J.G. (1991) Embryonic stem cells lacking a functional inhibitory G-protein subunit (αi2) produced by gene targeting of both alleles.Proc. Natl. Acad. Sci. USA 88, 7036–40.
Paris, S. and Pouysségur, J. (1986) Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts.EMBO J. 5, 55–60.
Pennington, S.L. and Wilson, J.H. (1991) Gene targeting in Chinese hamster ovary cells is conservative.Proc. Natl Acad. Sci. USA 88, 9498–502.
Ramírez-Solis, R., Rivera-Pérez, J., Wallace, J.D., Wims, M., Zheng, H. and Bradley, A. (1992) Genomic DNA microextraction: a method to screen numerous samples.Anal. Biochem. 201, 331–5.
Riele, H. te., Robanus Maandag, E., and Berns, A. (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs.Proc. Natl. Acad. Sci. USA 89, 5128–32.
Robertson, E.J. (1987) Embryo-derived stem cell lines. In Robertson, E.J. ed.,Teratocarcinomas and Embryonic Stem Cells: a Practical Approach pp. 71–112. Oxford: IRL Press Ltd.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual, 2nd edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Soriano, P., Montgomery, C., Geske, R. and Bradley, A. (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in miceCell 64, 693–702.
Tang, W.J. and Gilman, A.G. (1992) Adenylyl cyclases.Cell 70, 869–72.
Thomas, K.R. and Capecchi, M.R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell 51, 503–12.
Thomas, K.R., Deng, C. and Capecchi, M. (1992) High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors.Mol. Cell. Biol. 12, 2919–23.
Valancius, V. and Smithies, O. (1991) Double-strand gap repair in a mammalian gene targeting reaction.Mol. Cell. Biol. 11, 4389–97.
Wong, Y.H., Federman, A., Pace, A.M., Zachary, I., Evans, T., Pouysségur, J. and Bourne, H.R. (1991) Mutant α subunits of Gi2 inhibit cyclic AMP accumulation.Nature 351, 63–5.
Yang, L., Baffy, G., Rhee, S.G., Manning, D., Hansen, C.A. and Williamson, J.R. (1991) Pertussis toxin-sensitive G protein involvement in epidermal growth factor-induced activation of phospholipase C-γ in rat hepatocytes.J. Biol. Chem. 266, 22451–58.
Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A.M. and Birnbaumer, L. (1988) The G protein-gated atrial K+ channel is stimulated by three distinct Giα-subunits.Nature 336, 680–2.
Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. and Jaenisch, R. (1989) Germ-line transmission of a disrupted β2-microglobulin gene produced by homologous recombination in embryonic stem cells.Nature 342, 435–8.
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Rudolph, U., Brabet, P., Hasty, P. et al. Disruption of the Gi2α locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair. Transgenic Research 2, 345–355 (1993). https://doi.org/10.1007/BF01976176
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DOI: https://doi.org/10.1007/BF01976176