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

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

Research output: Contribution to journalArticle

60 Citations (Scopus)

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
Externally publishedYes

Fingerprint

Embryonic Stem Cells
Clone Cells
Genes
Genetically Modified Animals
Totipotent Stem Cells
In Vitro Techniques
Complementary DNA
Alleles
Galactosidases
vpr Genes
Alternative Splicing
Chondrocytes
Neuroglia
Muscle Cells
In Situ Hybridization
Cell Differentiation
Neurons
Messenger RNA

ASJC Scopus subject areas

  • Developmental Biology

Cite this

In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse. / Baker, Robert K.; Haendel, Melissa; Swanson, Bradley J.; Shambaugh, Janet C.; Micales, Bruce K.; Lyons, Gary E.

In: Developmental Biology, Vol. 185, No. 2, 15.05.1997, p. 201-214.

Research output: Contribution to journalArticle

Baker, Robert K. ; Haendel, Melissa ; Swanson, Bradley J. ; Shambaugh, Janet C. ; Micales, Bruce K. ; Lyons, Gary E. / In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse. In: Developmental Biology. 1997 ; Vol. 185, No. 2. pp. 201-214.
@article{9d95eea0e3554acc86e975f0db85c4fd,
title = "In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse",
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.",
author = "Baker, {Robert K.} and Melissa Haendel and Swanson, {Bradley J.} and Shambaugh, {Janet C.} and Micales, {Bruce K.} and Lyons, {Gary E.}",
year = "1997",
month = "5",
day = "15",
doi = "10.1006/dbio.1997.8541",
language = "English (US)",
volume = "185",
pages = "201--214",
journal = "Developmental Biology",
issn = "0012-1606",
publisher = "Academic Press Inc.",
number = "2",

}

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

AU - Swanson, Bradley J.

AU - Shambaugh, Janet C.

AU - Micales, Bruce K.

AU - Lyons, Gary E.

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.

UR - http://www.scopus.com/inward/record.url?scp=0030926713&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030926713&partnerID=8YFLogxK

U2 - 10.1006/dbio.1997.8541

DO - 10.1006/dbio.1997.8541

M3 - Article

VL - 185

SP - 201

EP - 214

JO - Developmental Biology

JF - Developmental Biology

SN - 0012-1606

IS - 2

ER -