Studies On Producing Chicken Chimeras Using BCs And Its Application

Based on the traditional color model for chicken chimeras, the multi-phenotype chimeric model was brought forward using the black silkies chicken with 10 characters. The way to produce chicken chimeras is improved greatly as a result of studies on the separating and dissociating of blastoderm, windowing the egg on equatorial plane, hatching without host eggshells, as well as transferring of the donor BCs. A practical approach was proposed to produce transgenic chicken chimeras using BCs, lipofectin and reporter gene (GFP). Furthermore, because of the problems in testing of germline chimeras and the relationship with homotypic chimeras, the application of W chromosome in these fields was discussed.The results indicated: (1)Apart from the black color, the silky feather was able to be used as phenotype marker in chicken chimeric model. However, it was difficult to get silky chimeras. (2) The way of producing chicken chimeras by BCs was improved, such as windowing on equatorial plane, sealing with the part of eggshell cut before as well as hatching without host eggshell et al. The highest hatchability was 39.7% (29/73) , the ratio of alive chimeras was 17.8% (13/73 ) as well as the ratio of chimeric embryos was 26.0% (19/73) . Apart from black feather, some other characters among the ten special phenotypes owned only by donor were found in many chimeric chickens. (3)To study germline chimeras, the cross of chimeras x donor hens was performed. 4 out of 7 chimeric roosters- No. 6, 8, 11, 12 produced offspring with black feather as a rate of 2.5% ( 3/118 ) ,31.6% ( 6/19 ) ,71.4%(15/21 ) and 33.3% ( 18/54 ) respectively. Especially, 3 chimeric roosters-No.6, 8, 12 produced offspring with black silky feather as a rate of 1.7%(2/118),10.5%( 2/19) ,1.8% ( 1/54 ) respectively. In the cross of chimeras x donor ,the rate of offspring with black feather and black silky feather is 29.4% ( 35/119 ) ,1.7% ( 2/119 ) respectively.(4) The blastoderm was separated and dispersed mechanically. The BCs from 4 blastoderms were cultured in vitro in a 35mm petridish with growth medium. Each petridish was used as one unit of transfection. The pEGFP-Cl plasmid and LIPOFECTAMINETM Reagent were transfect the BCs cultured in vitro for 4 hours. The following is the optimized condition: 1.5ug plasmid DNA, 5uL lipofectamine (1mg/mL), 30min to form the complex of DNA-liposome, 6h for incubating the cells with the complexes, 12h after adding medium contained serum. There are 30% cells, which transfected in vitro, emitted green light under fluorescent microscope. However, it was unable to get the positive chimeric embryos, which shine with green light at the moment. (5) It was a promising way in determination of the sex of the blastderm used in homotypic chimeras producing as well as in testing of germline chimeras to use the 447bp band of the W chromosome amplified by PCR.