TY - JOUR
T1 - Determination of transgenic loci by expression FISH
AU - Fan, Yuhong
AU - Braut, Steven A.
AU - Lin, Qingcong
AU - Singer, Robert H.
AU - Skoultchi, Arthur I.
N1 - Funding Information:
We thank Dr. Raju Kucherlapati for helpful discussions. This work was supported by National Institutes of Health grants to A.I.S. (CA 79057) and to R.H.S. (GM 54887).
PY - 2001/1/1
Y1 - 2001/1/1
N2 - 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.
AB - 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.
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U2 - 10.1006/geno.2000.6403
DO - 10.1006/geno.2000.6403
M3 - Article
C2 - 11161798
AN - SCOPUS:0035140344
SN - 0888-7543
VL - 71
SP - 66
EP - 69
JO - Genomics
JF - Genomics
IS - 1
ER -