Production of Chick Germline Chimeras from Fluorescence-Activated Cell-Sorted Gonocytes

Abstract

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.

Characteristics of Long-Term Cultures of Avian Primordial Germ Cells and Gonocytes1

“…Germ cells freshly isolated from the gonad have been shown previously to contribute to the germline after injection into recipient embryos [23,24]. This study shows that these gonocytes can retain their potential in culture and contribute to the germline.…”

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken1

“…3). This level of PGC purity is equal to those achieved with the FACS [4] and Nycodenz [8] methods. The red blood cells disappeared and the PGCs appeared when isolated whole blood was cultured for 1-2 weeks [19].…”

“…ACK lysis buffer consisted of 150 mM NH 4 Cl, 1 mM KHCO 3 , and 0.001 mM EDTA. The buffer was filter-sterilized through a 0.2-lm filter, and the prepared blood sample was added to 900 ll of ACK lysis buffer.…”

“…The chick PGCs migrate into the germinal ridge [1][2][3][4], where they differentiate into either spermatogonia in the testis or oogonia in the ovary. The unique migratory pathway of avian PGCs facilitates their isolation and transfer, especially from the blood vessels.…”

Primordial germ cells (PGCs) as a tool for creating transgenic chickens

“…Naito et al (2004) demonstrated that PGCs isolated from blood, and transferred to blastoderms in X stage, could enter in the bloodstream and migrate to the gonadal ridges of recipient embryos. Mozdziak et al (2006) used FACS to enrich PGC populations from gonocytes. Cell preparations obtained from male gonads from 10-14 embryos at stage 27 were incubated with a monoclonal antibody against SSEA-1, and fluorescently labeled cells were separated from non-reactive cells using FACS.…”