Advertisement

Transgenic Research

, Volume 6, Issue 1, pp 97–106 | Cite as

A molecular strategy designed for the rapid screening of gene traps based on sequence identity and gene expression pattern in adult mice

  • Donald Holzschu
  • Lorie Lapierre
  • Deborah Neubaum
  • Willie H. Mark
Article

Abstract

We have devised a strategy to rapidly screen gene traps in mouse embryonic stem (ES) cells based on DNA sequence information and an in vitro analysis of gene expression. After the initial identification of ES cell clones expressing β-galactosidase, tagged RNA transcripts were immediately cloned and sequenced in order to determine their identities. Novel gene sequences found were used to probe northern blots to examine the expression patterns of their cognate genes. Our initial characterization of 30 cDNA clones indicated that more than half of the tagged sequences were novel mouse genes and of these 40% showed a restricted pattern of expression in adult mouse tissues. This molecular characterization of gene traps is quick, reliable and well suited for the large-scale screening of mammalian developmental genes. Furthermore, since gene trap insertion frequently disrupts the tagged host gene, the ES cells can be used to produce transgenic animals for a genetic analysis of gene function

retroviral insertions promoter tagging cDNAcloning EST differential gene expression 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adams, M.D., Kelley, J.M., Gocayne, J., Dubnick, M., Polymeropoulos, M., Xiao, H., Merril, C., Wu, A., Olde, B., Moreno, R.F., Kerlavage, A.R., McCombie, W.R. and Venter, J.C. (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651-6.Google Scholar
  2. Adams, M.D., Dubnick, M., Kerlavage, A., Moreno, R., Kelley, J., Utterback, T., Nagle, J., Fields, C. and Venter, J.C. (1992) Sequence identification of 2,375 human brain genes. Nature 355, 632-4.Google Scholar
  3. Adams, M.D., Kerlavage, A., Fields, C. and Venter, J.C. (1993a) 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet. 4, 256-67.Google Scholar
  4. Adams, M.D., Soares, M.B., Kerlavage, A., Fields, C. and Venter, J.C. (1993b) Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nature Genet. 4, 373-80.Google Scholar
  5. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-10.Google Scholar
  6. Antequera, F. and Bird, A. (1993) Number of CpG islands and genes in human and mouse. Proc. Natl Acad. Sci. USA 90, 11995-9.Google Scholar
  7. Bier, E., Vaessin, H., Shepherd, S., Lee, K., McCall, K., Barbel, S., Ackerman, L., Carretto, R., Uemura, T., Grell, E., Jan, L.Y. and Jan, Y.N. (1989) Searching for pattern and mutation in the Drosophilagenome with a P-lacZ vector. Genes Dev. 3, 1273-87.Google Scholar
  8. Chen, Z., Friedrich, G.A. and Soriano, P. (1994) Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice. Genes Dev. 8, 2293-301.Google Scholar
  9. Copeland, N.G., Jenkins, N.A., Gilbert, D., Eppig, J.T., Maltais, L.J., Miller, J., Dietrich, W.F., Weaver, A., Lincoln, S., Steen, R.G., Stain, L., Nadeau, J.H. and Lander, E. (1993) A genetic linkage map of the mouse: current applications and future prospects. Science 262, 57-66.Google Scholar
  10. Coulombe, B., Ponton, A., Daigneault, L., Williams, B. and Skup, D. (1988) Presence of transcription regulatory elements within an intron of the virus-inducible murine TIMP gene. Mol. Cell. Biol. 8, 3227-34.Google Scholar
  11. DeGregori, J., Russ, A., von Melchner, H., Rayburn, H., Priyaranjan, P., Jenkins, N.A., Copeland, N.G. and Ruley, H.E. (1994) A murine homolog of the yeast RNA1 gene is required for postimplantation development. Genes Dev. 8, 265-76.Google Scholar
  12. Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R. (1985) The in vitrodevelopment of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morph. 87, 27-45.Google Scholar
  13. Edwards, D., Waterhouse, P., Holman, M. and Denhardt, D.T. (1986) A growth-responsive gene (16C8) in normal mouse fibroblasts homologous to human collagenase inhibitor with erythroid-potentiating activity: evidence for inducible and constituitive transcripts. Nucl. Acids Res. 14, 8863-78.Google Scholar
  14. Fathi, Z., Corjay, M., Shapira, H., Wada, E., Benya, R., Jensen, R., Viallet, J., Sausville, E. and Battey, J. (1993) BRS-3: A novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J. Biol. Chem. 268, 5979-84.Google Scholar
  15. Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13.Google Scholar
  16. Fields, C., Adams, M., White, O. and Venter, J.C. (1994) How many genes in the human genome. Nature Genet. 7, 345-6.Google Scholar
  17. Friedrich, G. and Soriano, P. (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513-23.Google Scholar
  18. Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl Acad. Sci. USA 85, 8998-9002.Google Scholar
  19. Gewert, D., Coulombe, B., Castelino, M., Skup, D. and Williams, B. (1987) Characterization and expression of a murine gene homologous to human EPA/TIMP: a virus-induced gene in the mouse Embo J. 6, 651-7.Google Scholar
  20. Glaser, R.L., Wolfner, M. and Lis, J. (1986) Spatial and temporal pattern of hsp26expression during normal development. EMBO J. 5, 747-54.Google Scholar
  21. Gorbulev, V., Akhundova, A., Grzeschik, K. and Fahrenholz, F. (1994) Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus. FEBS Lett. 340, 260-4.Google Scholar
  22. Gossler, A., Joyner, A.L., Rossant, J. and Skarnes, W.C. (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463-5.Google Scholar
  23. Green, M.C. (1989) Catalog of Mutant Genes and Polymorphic Loci. Gustav Fischer Verlag, Oxford: Oxford University Press.Google Scholar
  24. Gruber, A., Streit, A., Reist, M., Benninger, P., Boehni, R. and Schuemperli, D. (1990) Structure of a mouse histone-encoding gene cluster. Gene 95, 303-4.Google Scholar
  25. Haffner, C., Jarchau, T., Reinhard, M., Hoppe, J., Lohmann, S. and Walter, U. (1995) Molecular cloning, structural analysis, and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP. EMBO J. 14, 19-27.Google Scholar
  26. Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994) Manipulating the Mouse Embryo: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  27. Hoog, C. (1991) Isolation of a large number of novel mammelian genes by a differential cDNA library screening strategy. Nucl. Acids Rs. 19, 6123-6127.Google Scholar
  28. Horstrup, K., Jablonka, B., Hoenig-Liedl, P., Just, M., Kochsiek, K. and Walter, U. (1994) Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with f ibrinogen receptor inhibition. Eur. J. Biochem. 225, 21-7.Google Scholar
  29. Jain, R., Gomer, R.H. and Murtagh, J., Jr (1992) Increasing specificity from the PCR-RACE technique. BioTechniques 12, 58-9.Google Scholar
  30. Johnson, M. and Mahon, K.A. (1993) Detection of genes that are differentially expressed during mouse embryogenesis by genetic trapping strategies. Am. J. Obstet. Gynecol. 169, 683-9.Google Scholar
  31. Kerr, S.M., Vambrie, S., McKay, S.J. and Cooke, H.J. (1994) Analysis of cDNA sequences from mouse testis. Mamm. Genome 5, 557-65.Google Scholar
  32. Kontgen, F., Suess, G., Stewart, C., Steinmetz, M. and Bluethmann, H. (1993) Targeted disruption of the MHC class II AA gene in C57B1/6 mice. International Immunol 5, 957-64.Google Scholar
  33. Lion, T. and Haas, O. (1990) Nonradioactive labelling of probe with digoxigenin by polymerase chain reaction. Anal. Biochem. 18, 335-7.Google Scholar
  34. Macleod, D., Lovell-Badge, R., Jones, S. and Jackson, I. (1991) A promoter trap in embryonic stem (ES) cells selects for integration of DNA into CpG islands. Nucl. Acids Res. 19, 17-23.Google Scholar
  35. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  36. Markowitz, D., Goff, S. and Bank, A. (1988) A safe packaging cell line for gene transfer: separating viral genes on two different plasmids. J. Virol. 62, 1120-4.Google Scholar
  37. Meinecke, M., Geiger, J., Butt, E., Sandberg, M., Jahnsen, T., Chakraborty, T., Walter, U., Jarchau, T. and Lohmann, S. (1994) Human cyclic GMP-dependent protein kinase Ib overexpression increases phosphorylation of an endogenous focal contact-associated vasodilator-stimulated phosphoprotein without altering the thrombin-evoked calcium response. Molec. Pharm. 46, 283-290.Google Scholar
  38. Mori, S., Goto, K., Goto, F., Mutakami, K., Ohkawara, S. and Yoshinaga, M. (1994) Dynamic changes in mRNA expression of neutrophils during the course of acute inflammation in rabbits. Int. Immunol. 6, 149-156.Google Scholar
  39. Nakamura, H., Izumoto, Y., Kambe, H., Kuroda, T., Mori, T., Kawamura, K., Yamamoto, H. and Kishimoto, T. (1994) Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. J. Biol. Chem. 269, 25143-9.Google Scholar
  40. Niwa, H., Araki, K., Kimura, S., Taniguchi, S., Wakasugi, S. and Yamamura, K. (1993) An efficient gene-trap method using poly A trap vectors and characterization of gene-trap events. J. Biochem. 113, 343-9.Google Scholar
  41. Orr, S.L., Hughes, T., Sawyers, C.L., Kato, R., Quan, S., Williams, S., Witte, O. and Hood, L. (1994) Isolation of unknown genes from human bone marrow by differential screening and singlepass cDNA sequence determination. Proc. Natl Acad. Sci. USA 91, 11869-73.Google Scholar
  42. Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl Acad. Sci. USA 85, 2444-8.Google Scholar
  43. Skarnes, W.C., Auerbach, B.A. and Joyner, A. (1992) A gene trap approach in mouse embryonic stem cells: the lacZ reporter is activated by splicing, reflects endogenous gene expression, and it mutagenic in mice. Genes Dev. 6, 903-18.Google Scholar
  44. Takeda, J., Yano, H., Eng, S., Zeng, Y. and Bell, G. (1993) A molecular inventory of human pancreatic islets: sequence analysis of 1000 cDNA clones. Human Mol. Genet. 2, 1793-8.Google Scholar
  45. von Melchner, H., DeGregori, J., Rayburn, H., Reddy, S., Friedel, C. and Ruley, H.E. (1992) Selective disruption of genes expressed in totipotent embryonal stem cells. Genes Dev. 6, 919-27.Google Scholar
  46. Wilson, C., Pearson, R.K., Bellen, H.J., O'Kane, C.J., Grossniklaus, U. and Gehring, W.J. (1989) P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 3, 1301-13.Google Scholar
  47. Wurst, W., Rossant, J., Prideaux, V., Kownacks, M., Joyner, A., Hill, D., Guillemont, F., Gasca, S., Cado, D., Auerbach, A. and Ang, S.L. (1995) A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice. Genetics 139, 889-99.Google Scholar

Copyright information

© Chapman and Hall 1997

Authors and Affiliations

  • Donald Holzschu
    • 2
  • Lorie Lapierre
    • 2
  • Deborah Neubaum
    • 1
  • Willie H. Mark
    • 1
  1. 1.Section of Genetics & DevelopmentCornell UniversityIthacaUSA
  2. 2.Department of Microbiology and ImmunologyCollege of Veterinary Medicine, Cornell UniversityIthacaUSA

Personalised recommendations