[Chromosomal localization of foreign genes in transgenic mice using dual-color fluorescence in situ hybridization].
OBJECTIVE
To establish a highly sensitive and specific dual-color fluorescence in situ hybridization (D-FISH) method used for chromosomal localization of foreign genes in double transgenic mice.


METHODS
Two strains of double transgenic mice were used in this experiment, one was integrated with the herpes simplex virus thymidine kinase (HSV-tk) and the enhanced green fluorescence protein (eGFP), the other was with the short hairpin RNA interference(RNAi) and beta(654). Splenic cells cultured in vitro were arrested in metaphase by colchicine and hybridized with digoxigenin-labeled and biotinylated DNA probes, then detected by rhodamine-conjugated avidin and FITC-conjugated anti-digoxigenin.


RESULTS
Dual-color fluorescence signals were detected on the same metaphase in both transgenic mice strains. In HSV-tk/eGFP double transgenic mice, strong green fluorescence for HSV-tk and red for eGFP were observed and localized at 2E5-G3 and 8A2-A4 respectively. In beta(654)/RNAi mice, beta(654) was detected as red fluorescence on chromosome 7D3-E2, and RNAi showed random integration on chromosomes. It was detected as green fluorescence on chromosome 12B1 in one mouse, while on 1E2.3-1F and 3A3 in the other.


CONCLUSIONS
Highly sensitive and specific D-FISH method was established using the self-prepared DNA probes, and chromosomal localization of the foreign genes was also performed in combination with G-banding in double transgenic mice. This technology will facilitate the researches in transgenic animals and gene therapy models.
Lin
Dan
D
Ministry of Health, Institute of Shanghai Medical Genetics, Shanghai Jiaotong University, Shanghai, People's Republic of China.
Gong
Xiu-li
XL
Li
Wei
W
Guo
Xin-bing
XB
Zhu
Yi-wen
YW
Huang
Ying
Y
D000818:Animals; D002478:Cells, Cultured; D003116:Color; D049452:Green Fluorescent Proteins / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D020161:Physical Chromosome Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D019076:Transgenes
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase