Elsevier

Developmental Biology

Volume 185, Issue 2, 15 May 1997, Pages 201-214
Developmental Biology

Regular Article
In VitroPreselection of Gene-Trapped Embryonic Stem Cell Clones for Characterizing Novel Developmentally Regulated Genes in the Mouse

https://doi.org/10.1006/dbio.1997.8541Get rights and content
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Abstract

We have developed anin vitrogene 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 selectablelacZ/neoRfusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiatein 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 situhybridization analyses show that trapped genes are expressedin vivowithin the cell types that express β-galin 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 bylacZ/neoRin the hybrid gene trap transcript. This approach, which we term “in vitropreselection,” is more economical than standardin vivogene 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 differentiationin vitroas a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types.

Cited by (0)

A. L. Joyner, Ed.

1

The first three authors contributed equally to the manuscript.

2

To whom correspondence should be addressed at Department of Anatomy/318 SMI, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706. Fax: (608) 262-7306. E-mail: [email protected].