The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.
Part 1: Pregnant Mouse Laparotomy
Part 2: Gonadal Ridge Dissection
Under stereomicroscope and Light source Schott Fostec
Part 3: Gonadal Ridge Digestion
Under stereomicroscope:
Part 4: Primordial Germinal Cells Culture
“You should perform the next steps quickly to preserve the viability of PGCs for isolation and derivation.
Part 5: Embryonic Germinal Cells
Notes
Conclusion
We have provided a video that shows you how to isolate, derive, and culture embryonic germinal cells from gonadal ridges of 10.5-11.5 dpc mouse embryos. The reproducible isolation and long term culture of Embryonic germinal cell lines provides a critical foundation for study of early embryonic development, genetic and epigenetic germinal patterns, gonad formation, environmental effects of surrounding organs during the developmental process of gametogenesis in the male and female embryos, and determination of pathways that leads to cancer and infertility.
The authors have nothing to disclose.
The authors would like to acknowledge the invaluable help of: Dr Neal First and Kourtney Wilkinson for manuscript editing; Dr Lucy Senter, Dr. Brigit Willeford and Mike Basett for assistance with training and animal care at the MSU ALAC accredited mouse facility; Dr Dwayne Wise for his assistance with microscopy and image capture; Cesar Monroy, Hannah Swoope, and Bobbie Huddleston for their assistance with video production at MSU. This research was funded by Office of Institutional Research and the Department of Biological Sciences at Mississippi State University.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
10.5 – 11.5 dpc pregnant C57BL6J female mouse crossed with the same background male mouse. | ||||
Mouse Fibroblast MEF-CF1 ATCC | SCRC-1040 | |||
Dulbecco’s Phosphate Buffered Saline (D-PBS) | Invitrogen | 14190/086 | ||
Collagenase /Dispase Solution | Roche | 269638 | ||
Gelatin | Sigma | G1890 | ||
Mitomycin C | Sigma | M4287 | ||
Trypsin EDTA | Invitrogen | 25200-072 | ||
MEF Medium: Dulbecco Medium Eagle Modified (D-MEM) GLUTAMAX High glucose | Invitrogen | 10566024 | ||
10% Fetal Bovine Serum | Invitrogen | 16000/044 | ||
1% of antibiotic-antimycotic | Invitrogen | 15420/096 | ||
Knockout Media: 80% knockout D-MEM | Invitrogen | 10829/018 | ||
20% Knockout serum Invitrogen Cat. No. 1028/028, 200mM L-glutamine Invitrogen | 25130/081 | with β-mercaptoethanol Sigma Catalogue Number: M7522 | ||
1X non essential amino acid solution | Invitrogen | 11140/050 | ||
1% antibiotic-antimycotic | Invitrogen | 15420/096 | supplemented with grow factor as 2500 U of Leukemia inhibitory factor (mLIF-ESGRO) Millipore Catalogue Number: ESG1106 | |
40 ng/ml of Recombinant Murine Stem Cell Factor (SCF) ReproTech | 250-03 | |||
20 ng/ml of Basic Fibroblast Growth factor (bFGF) | Invitrogen | 13256-029 | ||
Corning center well culture dish 60mm | Fisher | 07-200-79 | 1.5 ml eppendorf tube, and 15 ml falcon tubes |