MPSS profiling of embryonic gonad and primordial germ cells

ABSTRACT: We used the MPSS technology to uncover gene expression profiling in a greater depth in the early embryonic gonads and primordial germ cells (PGCs) in the chicken. Total numbers of sequenced signatures were 1,012,533 and 995,676 for the PGCs and gonad. Using a false discovery rate cut-off of 0.05, we found 3.05 % of all signatures in the PCGs and 1.89 % of all signatures in the gonad signatures were significantly up-regulated compared to each sample. The MPSS result was very consistent with our previous result using EST data(http://chickgce.snu.ac.kr/). Keywords: cell type comparison Experimental animals provided for this experiment were maintained at the University Animal Farm, Seoul National University, and all experimental procedures were performed at the affiliated laboratories of the university. Gonadal cells were retrieved from the gonads of 6.5-day-old (stage 29) White Leghorn (WL) embryos by our standard procedure [26]. Embryos were freed from the yolk by rinsing with calcium- and magnesium-free PBS and the gonads were retrieved by dissection of embryo abdomen with sharp tweezers under a stereomicroscope. Embryonic gonads were collected from total 1,947 embryos by 10 highly-skilled persons through 8 separated experimental batches. Gonadal tissues were dissociated by gentle pipetting in 0.05% (v:v) trypsin solution supplemented with 0.53 mM EDTA. After centrifuged at 200xg for 5 min, total gonadal cells were loaded into MACS (Miltenyi Biotech, Germany), and the separated primordial germ cells (PGCs) were immediately stored in liquid nitrogen (-190ºC) until processed further. The numbers of PGCs in cell population before and after loading were counted. MACS treatment for chicken PGCs and counting PGC number Chicken gonadal cells were incubated with PGC-specific primary antibody, anti-stage specific embryo antigen (anti-SSEA)-1 antibody for chicken PGCs (mouse IgM isotype), for 20 min at the room temperature of 20-25?C. Anti-SSEA-1 antibody developed by [27] was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Development of Biological Science. After washing with 1mL buffer (PBS supplement with 0.5% BSA and 2mM EDTA), the supernatant was completely removed. The pellet was mixed with 100 ? buffer containing 20? of rat anti-mouse IgM microbeads for 15 min at 4?C. Treated cells were carefully washed by the addition of 500 ? buffer and subsequently loaded with MACS.[28] For counting cell number, chicken PGCs before or after MACS treatment were fixed with 1% (v:v) glutaraldehyde for 5 min and rinsed with 1x PBS twice. The anti-SSEA-1 ascites fluid diluted 1:1,000 in PBS was added and subsequent steps were carried out using DAKO universal LSAB® kit, Peroxidase (DAKO, USA) according to the manufacturer’s instruction. After 8 batches of cell preparation, total cell number of PGC-enriched fraction and gonadal stromal cells was 5.26X106 and 1.76X108, respectively. These cell populations were further used for total RNA isolation and massively parallel signature sequencing (MPSS) analysis.

ORGANISM(S): Gallus gallus

SUBMITTER: heebal kim 

PROVIDER: E-GEOD-5909 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress