Determination of transgenic loci by expression FISH

Yuhong Fan, Steven A. Braut, Qingcong Lin, Robert H. Singer, Arthur I. Skoultchi

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

DNA targeting by homologous recombination in mouse embryonic stem (ES) cells has become a widely used method for manipulating the mouse genome and for studying the role of specific genes in mammalian development. For certain studies, it is necessary to target two or more DNA sequences residing on a particular chromosome. In these situations, it would be important to distinguish whether two sequential gene targeting events in the ES cells have occurred in cis or in trans. We report here a new application of fluorescence in situ hybridization to RNA molecules present at sites of transcription that allows the identification of cis and trans gene targeting events in ES cells. The method is based on detection of transcripts from commonly used selectable marker genes inserted during homologous recombination. Transcripts are detected in interphase nuclei, making the preparation of mitotic cells unnecessary and obviating the necessity for the more technically demanding DNA detection of genes. The method is applicable to any chromosomal locus, and compared with other methods (e.g., genetic linkage testing in chimeric mice), it will greatly shorten the time required for distinguishing cis and trans gene targeting events in ES cells. The method also may be useful for detecting changes in ploidy of individual chromosomes and loss of heterozygosity of genes in single cells in culture and also in animals, for example, during processes such as tumorigenesis.

Original languageEnglish (US)
Pages (from-to)66-69
Number of pages4
JournalGenomics
Volume71
Issue number1
DOIs
StatePublished - Jan 1 2001

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Gene Targeting
Embryonic Stem Cells
Homologous Recombination
Genes
Chromosomes
Genetic Linkage
Ploidies
Loss of Heterozygosity
DNA
Interphase
Genetic Testing
Fluorescence In Situ Hybridization
Carcinogenesis
Cell Culture Techniques
Genome
RNA

ASJC Scopus subject areas

  • Genetics

Cite this

Determination of transgenic loci by expression FISH. / Fan, Yuhong; Braut, Steven A.; Lin, Qingcong; Singer, Robert H.; Skoultchi, Arthur I.

In: Genomics, Vol. 71, No. 1, 01.01.2001, p. 66-69.

Research output: Contribution to journalArticle

Fan, Yuhong ; Braut, Steven A. ; Lin, Qingcong ; Singer, Robert H. ; Skoultchi, Arthur I. / Determination of transgenic loci by expression FISH. In: Genomics. 2001 ; Vol. 71, No. 1. pp. 66-69.
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