Transgene integration in hair follicles and peripheral blood cells measured by in vitro DNA amplification and fluorescence in situ hybridization.
Screening of animals to detect the presence of integrated DNA sequences is an essential component of transgenic mouse generation. Rapid and sensitive detection techniques to facilitate identification of transgenic animals for biological studies or subsequent breeding programs are desirable. Most transgenics are generated on F1 backgrounds, thus determination of the histocompatibility status of neonates provides important diagnostic information for establishing congenic colonies. We describe the application of two assays, in vitro DNA amplification using the polymerase chain reaction (PCR) and fluorescence in situ hybridization with biotinylated DNA probes, to facilitate rapid detection of transgenes and their chromosomal integration patterns in young mice. A noninvasive PCR-based assay to detect the transgene in DNA contained in detergent-extracted hair follicles was developed for rapid screening. A total of 147 mice derived from F2, F3, and F4 generations of C57BL x F1 (globin transgenics) were assayed to determine whether they carried a globin transgene. Characterization of animals by PCR-based amplification of the transgene was compared with that obtained using standard Southern analysis of DNA extracted from tails. Categorization of animals as positive (carrying the transgene) or negative using PCR was performed successfully in the initial assay with 95% of the animals. Fluorescence in situ hybridization with a DNA probe showing homology with a portion of the transgene was performed on metaphase and interphase cells to determine the integration pattern of the transgene. Our data showed that the transgene was integrated in a single chromosome. These techniques should facilitate rapid identification of transgenic animals and characterization of the genomic transgene integration patterns.
Amendola
R
R
Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550.
Haendel
S
S
Weier
H U
HU
Pallavicini
M G
MG
D000818:Animals; D001483:Base Sequence; D001710:Biotin; D001774:Blood Chemical Analysis; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004587:Electrophoresis, Agar Gel; D005453:Fluorescence; D005914:Globins / Q000235:genetics; D006197:Hair / Q000737:chemistry*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D001710:Biotin; D005914:Globins; D004247:DNA