Isolation of chicken primordial germ cells using fluorescence-activated cell sorting

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Abstract
Presently, it is difficult to undertake germ line modification of the chicken with primordial germ cells (PGC) because it has been difficult to efficiently fractionate the PGC from the total somatic cell population. The objective of this study was to develop a method that allows isolation of an enriched population of viable PGC from embryonic blood and embryonic gonadal tissue. Blood was harvested from early chick embryos (stages 13 to 15), and cells were liberated from the gonads of stage 27 chick embryos. Subsequently, viable PGC were labeled with anti-stage-specific embryonic antigen-1 (SSEA-1), which was detected with goat-anti-mouse IgM-fluorescein isothiocyanate. Fluorescently labeled cells were sorted from the unlabeled cells using fluorescence-activated cell sorting (FACS), and the identities of the PGC were confirmed using periodic acid-Schiff (PAS) staining or anti-embryonic mouse antigen-1 (EMA-1) staining followed by microscopic evaluation. Finally, PGC were sorted from somatic cells of sex-identified embryos. Less than 0.1% of the blood cell population was collected as SSEA-1-positive cells. Similarly, approximately 2% of the gonadal cell population were collected as SSEA-1-positive cells. Therefore, fewer (-1,000 to 9,000) PGC were recovered from each isolate. Placing the sorted SSEA-1-positive cells on a glass slide from a microcentrifuge tube resulted in a recovery rate of 53 to 73% relative to the number detected by FACS. Furthermore, the proportions of sorted cells that stained with PAS or anti-EMA-1 following sorting were 92+/-4% PAS positive and 94+/-1% anti-EMA-1 positive. Finally, the sorted SSEA-1-positive cells were maintained in vitro to demonstrate their viability after sorting. It was demonstrated that it is possible to label blood and gonadal chicken PGC with SSEA-1 and subsequently to sort viable SSEA-1-positive PGC from somatic cells.

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