title: Rapid localization of transgenes in mouse chromosomes with a combined Spectral Karyotyping/FISH technique.
abstract: We explored the feasibility of combined Spectral Karyotyping (SKY) and Fluorescence In Situ Hybridization (FISH) as means to rapidly map a chromosomally integrated renin/green fluorescent protein (GFP) fusion gene construct (Ren-GFP) in the transgenic mouse, Tg(Ren-GFP)1Kwg. A sequential hybridization with SKY probes followed by FISH gave consistently satisfactory results, demonstrating that multiple copies of the Ren-GFP transgene in this transgenic mouse line are integrated into a single chromosomal site of Chromosome (Chr) 4, most probably in the juxta-centromeric euchromatic region consisting of the A2-A3 domain. Chr 4 as a sole carrier of the transgene also was confirmed by co-hybridization to p1 BAC clone DNA containing telomeric sequences specific for mouse Chr 4 and the Ren-GFP construct in pGEM4Z vector. The hemizygosity of the Ren-GFP transgene is maintained not only in bone marrow cells, but also in lung cells proliferating in vitro, indicating that stable integration of the Ren-GFP transgene into chromosomal DNA was established at a very early embryonic stage. We conclude that the SKY/FISH technique is a reliable and facile method for establishing the integration site of a transgene. As such, this protocol has obvious advantages over traditional backcross methods in terms of time, cost and labor for determining the chromosomal location of transgenes.
authors.affiliation: Dept. of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. seiich.matsui@roswellpark.org
authors.forename: Sei-ichi
authors.initials: S
authors.lastname: Matsui
authors.forename: Sheila
authors.initials: S
authors.lastname: Sait
authors.forename: Craig A
authors.initials: CA
authors.lastname: Jones
authors.forename: Norma
authors.initials: N
authors.lastname: Nowak
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Gross
mesh_terms: D000818:Animals; D001158:Arteries / Q000166:cytology; D015139:Blotting, Southern; D001854:Bone Marrow Cells; D002455:Cell Division; D002875:Chromosomes / Q000235:genetics*; D003433:Crosses, Genetic; D005260:Female; D018628:Gene Dosage; D049452:Green Fluorescent Proteins; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics*; D008168:Lung / Q000166:cytology; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012083:Renin / Q000235:genetics*; D032681:Spectral Karyotyping / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012083:Renin