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

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.

Isolation of teleost primordial germ cells using flow cytometry

“…In mammals and birds, successful isolation of PGCs by MACS was achieved using antibodies against cell surface markers expressed on the PGCs (Pesce and De Felici, 1995;Durcova-Hills et al, 1999-2000Mozdziak et al, 2005). A small number of PGC cell surface markers are available for producing useful antibodies for this system.…”

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

“…However, an efficient isolation method for cPGCs has not been established to date. The main methods used to purify avian cPGCs are Ficoll density gradient centrifugation [5], Nycodenz density gradient centrifugation [8], immunomagnetic cell sorting (MACS) [9], and fluorescence-activated cell sorting (FACS) [10]. However, these techniques require special training and are quite laborintensive.…”

The Evaluation of Various Conditions in the Cryopreservation of Primordial Germ Cells on Korean Native Chicken (Ogye)

“…본 연구와 동일한 발생 단계인 생식선 유래의 gonadal PGC(gPGC)를 이용한 연구 (Natio et al, 1994;Natio, 2003;Zhao and Kuwana, 2003;Mozdziak et al, 2005) (Fig. 3A) 그리고 한국육종협회3호종 (Fig.…”