Enrichment and efficient screening of ES cells containing a targeted mutation: The use of DT-A gene with the polyadenylation signal as a negative selection maker

Yuchio Yanagawa, Takashi Kobayashi, Motoko Ohnishi, Takayasu Kobayashi, Shinri Tamura, Teruhisa Tsuzuki, Makoto Sanbo, Takeshi Yagi, Fumi Tashiro, Jun ichi Miyazaki

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DT-A), has been commonly used. In this study, we developed a negative selection marker using DT-A gene with polyadenylation signal and it was designated DT-ApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3' end, second, TK at both the 5' and 3' ends <2 XTK>, and third, DT-ApA at the 5' end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DT-ApA was more efficient than that using TK for homologous recombination and that negative selection using DT-ApA was as efficient as that using 2 X TK. Considering the fact that the use of DTApA is more convenient for construction of targeting vectors than that of 2 X TK, DT-ApA is an efficient negative selection marker. In addition, we examined long and accurate PCR (LA-PCR) for screening gene targeted clones. The use of LAPCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LA-PCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DT-ApA for negative selection together with the application of LA-PCR for screening ensures efficient and time-saving screening for homologous recombinants.

Original languageEnglish
Pages (from-to)215-221
Number of pages7
JournalTransgenic Research
Volume8
Issue number3
DOIs
Publication statusPublished - 1999 Jun 1

Keywords

  • Diphtheria toxin
  • Gene targeting
  • LA-PCR
  • Negative selection
  • Polyadenylation signal
  • Screening

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Agronomy and Crop Science
  • Genetics

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