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. 1990 Nov 11;18(21):6247–6251. doi: 10.1093/nar/18.21.6247

Generation of small mutation in large genomic fragments by homologous recombination: description of the technique and examples of its use.

M Tripodi 1, S Perfumo 1, R Ali 1, L Amicone 1, C Abbott 1, R Cortese 1
PMCID: PMC332488  PMID: 2243773

Abstract

We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ciliberto G., Dente L., Cortese R. Cell-specific expression of a transfected human alpha 1-antitrypsin gene. Cell. 1985 Jun;41(2):531–540. doi: 10.1016/s0092-8674(85)80026-x. [DOI] [PubMed] [Google Scholar]
  2. D'Onofrio C., Colantuoni V., Cortese R. Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals. EMBO J. 1985 Aug;4(8):1981–1989. doi: 10.1002/j.1460-2075.1985.tb03881.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. De Simone V., Ciliberto G., Hardon E., Paonessa G., Palla F., Lundberg L., Cortese R. Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene. EMBO J. 1987 Sep;6(9):2759–2766. doi: 10.1002/j.1460-2075.1987.tb02570.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  7. Kidd V. J., Wallace R. B., Itakura K., Woo S. L. alpha 1-antitrypsin deficiency detection by direct analysis of the mutation in the gene. Nature. 1983 Jul 21;304(5923):230–234. doi: 10.1038/304230a0. [DOI] [PubMed] [Google Scholar]
  8. Labeit S., Lehrach H., Goody R. S. DNA sequencing using alpha-thiodeoxynucleotides. Methods Enzymol. 1987;155:166–177. doi: 10.1016/0076-6879(87)55015-7. [DOI] [PubMed] [Google Scholar]
  9. Long G. L., Chandra T., Woo S. L., Davie E. W., Kurachi K. Complete sequence of the cDNA for human alpha 1-antitrypsin and the gene for the S variant. Biochemistry. 1984 Oct 9;23(21):4828–4837. doi: 10.1021/bi00316a003. [DOI] [PubMed] [Google Scholar]
  10. Monaci P., Nicosia A., Cortese R. Two different liver-specific factors stimulate in vitro transcription from the human alpha 1-antitrypsin promoter. EMBO J. 1988 Jul;7(7):2075–2087. doi: 10.1002/j.1460-2075.1988.tb03047.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Poustka A., Rackwitz H. R., Frischauf A. M., Hohn B., Lehrach H. Selective isolation of cosmid clones by homologous recombination in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4129–4133. doi: 10.1073/pnas.81.13.4129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Povey S. Genetics of alpha 1-antitrypsin deficiency in relation to neonatal liver disease. Mol Biol Med. 1990 Apr;7(2):161–172. [PubMed] [Google Scholar]
  13. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  15. Whitehouse D. B., Abbott C. M., Lovegrove J. U., McIntosh I., McMahon C. J., Mieli-Vergani G., Mowat A. P., Hopkinson D. A. Genetic studies on a new deficiency gene (PI*Ztun) at the PI locus. J Med Genet. 1989 Dec;26(12):744–749. doi: 10.1136/jmg.26.12.744. [DOI] [PMC free article] [PubMed] [Google Scholar]

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