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1
GENE TRANSFER BY MANIPULATION OF PRIMORDIAL GERM CELLS IN THE CHICKEN

Han, Jac Y.,Shoffner, R.N.,Guise, K.S. Asian Australasian Association of Animal Productio 1994 Animal Bioscience Vol.7 No.3
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The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.
2
닭 초기 배아의 유전자 미세주입과 유전자 발현에 관한 연구

한재용(J . Y . Han),(R . N . Shoffner),(K . S . Guise) 한국축산학회 1994 한국축산학회지 Vol.36 No.3
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- 복사/대출신청
This study was carried out to examine the expression of marker genes and integration of plasmid DNA into the germ cells in young chicken embryos. The RSVLTR/βG2 plasmid contains the lacZ gene under the control of rous sarcoma virus (RSV) long terminal repeat(LTR) promoter. After a square window of 5㎜ per side was cut in the side of the egg with a dentistry drill, the transfection cocktail of calcium-phosphate or lipofectin with plasmid DNA was microinjected in the area of blastoderm and germinal crescent whose developmental stages were stage X and stage 6 to 8, respectively. Microinjected eggs were sealed, and the eggs were returned to the incubator with the $quot;window$quot; side up overnight. Microinjected embryos with plasmid DNA were screened with Xgal, and the marker gene was expressed in the brain, notochord and other parts of body of 1.5∼4.5 day old embryos, suggesting that developing stem cells in unincubated blastoderms or 1∼4 somite embryos can be transfected with plasmid vectors. The possibility of germ line integration with plasmid DNA by direct microinjection into early chicken embryos was determined. Transfection of stem cells for gonads in the blastoderm or germinal crescent with plasmid vectors was observed. Positive primordial germ cells in the gonad were not observed by plasmid DNA microinjection into unincubated or 24hr incubated embryos in this study. However, the expression of plasmid DNA with RSVLTR promoter in the early chicken embryonic cell shows the possibility of transgenic chicken production by direct microinjection with plasmid DNA. Also genetic manipulation of chicken production traits such as disease resistance, growth and production may he possible in the future.
3
STABLE TRANSFORMATION OF CULTURED CHICKEN CELLS

Han, J.Y.,Shin, Y.S.,Shoffner, R.N.,Guise, K.S. Asian Australasian Association of Animal Productio 1993 Animal Bioscience Vol.6 No.4
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A plasmid vector, $RSVLTR/{\beta}G2$, containing lacZ gene under the control of the RSVLTR promoter were transfected into chicken embryo fibroblasts by three different transfection methods. Calcium phosphate, lipsome and DEAE-dextran techniques were applied for transfection of chicken cells. A histochemical assay with X-gal was used as a simple method for screening transfected cells. Plasmid $RSVLTR/{\beta}G2$ was expressed proficiently in the chicken embryo fibroblast. Calcium phosphate-DNA precipitate transfection resulted in the highest efficiency for transient expression of $RSVLTR/{\beta}G2$. Transfected cells formed colonies on the 9th day of incubation indicating stable transformation of the inserted plasmid.