In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse

Robert K. Baker, Melissa A. Haendel, Bradley J. Swanson, Janet C. Shambaugh, Bruce K. Micales, Gary E. Lyons

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

We have developed an in vitro gene trap screen for novel murine genes that allows one to determine, prior to making chimeric or transgenic animals, if these genes are expressed in one or more specific embryonic tissues. Totipotent embryonic stem (ES) cells are infected with a retroviral gene trap construct encoding a selectable lacZ/neo(R) fusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiate in vitro, and neurons, glia, myocytes, and chondrocytes are screened for expression of β-galactosidase (β-gal). cDNAs of the gene trap transcripts are obtained by 5' rapid amplification of cDNA ends and are sequenced to determine if they represent novel genes. In situ hybridization analyses show that trapped genes are expressed in vivo within the cell types that express β-gal in vitro. Gene traps and their wild-type alleles are characterized in terms of copy number, alternate splicing of their transcripts, and the proportion of endogenous mRNA sequence that is replaced by lacZ/neo(R) in the hybrid gene trap transcript. This approach, which we term 'in vitro preselection,' is more economical than standard in vivo gene trap screening because tissue-specific expression of probable knockout alleles is verified before transgenic animals are generated. These results also highlight the utility of ES cell differentiation in vitro as a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types.

Original languageEnglish (US)
Pages (from-to)201-214
Number of pages14
JournalDevelopmental Biology
Volume185
Issue number2
DOIs
StatePublished - May 15 1997

ASJC Scopus subject areas

  • Molecular Biology
  • Developmental Biology
  • Cell Biology

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