Avian Karyoplasts in Inactivated Chicken Germ Stem Cells (GSCs): Avian Somatic Nuclear Transfer Facilitated by a Novel Antibody Alignment Method.

Abstract

The production of host chickens (Gallus gallus) with chimeric gonads producing gametes derived from transferred exotic donor-bird germ stem cells (GSCs), could potentially have a great impact on the management of threatened species. In this study a novel method of somatic nuclear transfer (snt) was developed using an antibody-biotin-avidin-antibody complex for alignment of somatic cells and GSCs for electrofusion. Quail GSC's were stained with the red fluorescent stains PKH-26 and ethidium bromide following irradiation with UV-light (250nm) for 60s creating an inactivated nucleus. Karyoplasts were produced by centrifugation of chicken fibroblasts at 10,000g on a 10% ficoll gradient and stained with the blue fluorescent stain Hoechst-33342. Inactivated GSCs and karyoplasts were stained with primary antibodies SSEA-1 (avian germ stem cell marker) and C1 (avian fibroblast specific marker), respectively. GSCs and karyoplasts were bound with secondary antibodies conjugated to biotin and avidin, respectively. To align the GSCs to karyoplasts, they were incubated at 37oc for 48 hours. The aligned cells were electrofused (two 2.5kV pulses at 10uF) and immediately resuspended in culture medium before fluorescent microscopy evaluation. Doublet cells were observed after incubation for up to 48 hours and following the first 2.5kV pulse. After the second 2.5kV fusion pulse, cells were observed to contain one each of a red and a blue nucleus, suggesting successful electrofusion of a single GSC and karyoplast. This method may be more efficient than random pearl chain formation for alignment of single GSCs and karyoplasts prior to electrofusion, providing a practical means of making the large numbers of snt-GSCs necessary for the production of chimeric gonads.