TY - JOUR
T1 - In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse
AU - Baker, Robert K.
AU - Haendel, Melissa A.
AU - Swanson, Bradley J.
AU - Shambaugh, Janet C.
AU - Micales, Bruce K.
AU - Lyons, Gary E.
N1 - Funding Information:
We thank Dr. Andras Nagy, Reka Nagy, Dr. Wanda Abramow-Newerly, and Dr. Janet Rossant for providing the R1 ES cells and Dr. Philippe Soriano for providing the ROSAβ-geo gene trap construct, gifts which made these experiments possible. We also thank Dr. Brigid Hogan for the E8.5 embryo cDNA library and Dr. Jeffrey Robbins for the differentiated ES cell cDNA library; Dr. Anthony Frankfurter for mouse anti-βIII-tubulin antibody; Dr. Miles Epstein for anti-GFAP antibody; and the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA) under Contract N01-HD-6-2915 from the NICHD for the other primary antibodies used. G.E.L. thanks Bradley Bodner, Joshua Daniels, Allen Last, and Dmitry Lapidus for their assistance in early stages of this research and Jayne Squirrell for her comments on the manuscript. We thank Dr. Peter Baas for the use of his confocal microscope to produce some of the images presented here. This work was supported by NIH Grant HD29471 to G.E.L., HD07342 and HL09584 to R.K.B., and GMO 7507-18 to M.A.H. and a grant from Goucher College to J.C.S. B.K.M. was supported in part by grants from the Muscular Dystrophy Association.
PY - 1997/5/15
Y1 - 1997/5/15
N2 - 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.
AB - 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.
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U2 - 10.1006/dbio.1997.8541
DO - 10.1006/dbio.1997.8541
M3 - Article
C2 - 9187083
AN - SCOPUS:0030926713
SN - 0012-1606
VL - 185
SP - 201
EP - 214
JO - Developmental Biology
JF - Developmental Biology
IS - 2
ER -