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Selection of transgenic preimplantation mouse embryos using fluorescence in situ hybridization
Theriogenology
39:258,
1993
SELECTION OF TRANSGENIC PREIMPLANTATION MOUSE EMBRYOS USING FLUORESCENCE -IN SITU HYBRIDIZATION
J. Lewls-Wllllams,T. Houseal’, M. Harvey, P. DlTulllo and C. Zlomek Geruyme Corporation, Framlngham, MA 01701 USA ‘IG Laboratories, Framlngham, MA 01701 USA The frequency of productlon of transgenlc (Tg) animals by oocyte mIcroInJectIon Is relatively high In mice (1O-30%), but lower (2- 10%) for llvestock species, Including goat, sheep, plg and bovlne. Therefore, a reliable method for the detection of Tg mIcroInJected embryos before their relmplantatlon In a foster mother Is highly deslrable. We have devised a protocol uslng fluorescence In situ hybrldlzatlon (FISH) for the ldentlflcatlon of mouse morula transgenlc for alpiiaT antltrypsln gene (BC30) as a model for optlmlzatlon of Tg goat productlon. Dlssoclated or blopsled blastomeres were treated wlth hypotonlc saline. flxed and alr dried on glass slides. The cells were cohybrldlzed wlth a dlgoxlgenln tagged probe for the transgene and a blotln tagged mouse genomlc probe as an internal control. Hybrlzatlon was vlsuallzed with FITC-anti dlgoxlgenln and Cy3-streptavldln. To test the feaslblllty of thls approach, dlssoclated or blopsled a-cell blastomeres were derived from a non-Tg female mated to a BC30 Tg male In which case we expect 50% of the embryos to be transgenlc. Average cell recovery was 76% (171/225 cells) and 94% (160/171) of those could be scored by FISH. Fifty percent (69/l 39) of the cells from dlssoclated embryos and 43% (9/21) of blopsled cells from lndlvldual embryos were scored as poslttve for the BC30 transgene. When cells from a single embryo were grouped, slmllar patterns of hybrldlzatlon were observed In all cells. To test thls approach directly, embryos were mlcrolnjected with BC30 at 1 ng/pI, cultured to the 8-lbcell stage In CZB medium, and dlssoclated to single cells In calcium free CZB medlum or blopsled. Twenty-SIX percent (9/34) of blopsled morula scored posltlve for the BC30 transgene. Average cell recovery was 66% (39/59) and 87% (34/39) of those could be scored by FISH. As a control for nonIntegrated DNA, 80 freshly Injected 1-cell embryos were also prepared for FISH. After processing, 68% of the l-cell pronuclel were recovered and the two pronuclel remained associated In 28% of embryos. In the latter cases, the larger male pronucleus displayed multiple or diffuse hybrldlzatlon signals for the transgene, while the smaller female pronucleus lacked hybrldlzatlon. In nelther case were discrete single posltlve hybridlzatlon slgnals detected. We conclude that fluorescence b $I& hybrldlzatlon can be used to detect transgenlc prelmplantatlon mouse embryos and we are currently adaptlng this methodology for use on the goat.
258
Copyright Q 1993 Butterworth-Heinemann
39:258,
1993
SELECTION OF TRANSGENIC PREIMPLANTATION MOUSE EMBRYOS USING FLUORESCENCE -IN SITU HYBRIDIZATION
J. Lewls-Wllllams,T. Houseal’, M. Harvey, P. DlTulllo and C. Zlomek Geruyme Corporation, Framlngham, MA 01701 USA ‘IG Laboratories, Framlngham, MA 01701 USA The frequency of productlon of transgenlc (Tg) animals by oocyte mIcroInJectIon Is relatively high In mice (1O-30%), but lower (2- 10%) for llvestock species, Including goat, sheep, plg and bovlne. Therefore, a reliable method for the detection of Tg mIcroInJected embryos before their relmplantatlon In a foster mother Is highly deslrable. We have devised a protocol uslng fluorescence In situ hybrldlzatlon (FISH) for the ldentlflcatlon of mouse morula transgenlc for alpiiaT antltrypsln gene (BC30) as a model for optlmlzatlon of Tg goat productlon. Dlssoclated or blopsled blastomeres were treated wlth hypotonlc saline. flxed and alr dried on glass slides. The cells were cohybrldlzed wlth a dlgoxlgenln tagged probe for the transgene and a blotln tagged mouse genomlc probe as an internal control. Hybrlzatlon was vlsuallzed with FITC-anti dlgoxlgenln and Cy3-streptavldln. To test the feaslblllty of thls approach, dlssoclated or blopsled a-cell blastomeres were derived from a non-Tg female mated to a BC30 Tg male In which case we expect 50% of the embryos to be transgenlc. Average cell recovery was 76% (171/225 cells) and 94% (160/171) of those could be scored by FISH. Fifty percent (69/l 39) of the cells from dlssoclated embryos and 43% (9/21) of blopsled cells from lndlvldual embryos were scored as poslttve for the BC30 transgene. When cells from a single embryo were grouped, slmllar patterns of hybrldlzatlon were observed In all cells. To test thls approach directly, embryos were mlcrolnjected with BC30 at 1 ng/pI, cultured to the 8-lbcell stage In CZB medium, and dlssoclated to single cells In calcium free CZB medlum or blopsled. Twenty-SIX percent (9/34) of blopsled morula scored posltlve for the BC30 transgene. Average cell recovery was 66% (39/59) and 87% (34/39) of those could be scored by FISH. As a control for nonIntegrated DNA, 80 freshly Injected 1-cell embryos were also prepared for FISH. After processing, 68% of the l-cell pronuclel were recovered and the two pronuclel remained associated In 28% of embryos. In the latter cases, the larger male pronucleus displayed multiple or diffuse hybrldlzatlon signals for the transgene, while the smaller female pronucleus lacked hybrldlzatlon. In nelther case were discrete single posltlve hybridlzatlon slgnals detected. We conclude that fluorescence b $I& hybrldlzatlon can be used to detect transgenlc prelmplantatlon mouse embryos and we are currently adaptlng this methodology for use on the goat.
258
Copyright Q 1993 Butterworth-Heinemann