Investigating Molecular Mechanisms of Embryonic Mammary Gland Development by Bead-Implantation in Embryonic Flank Explant Cultures – A Protocol

For flank explant cultures of older embryos Voutilainen et al. do not gelatinize the filters, but store them untreated in 70 % ethanol at room temperature and wash in DPBS just before use [7]. Although gelatinization of filters and storage in serum may provide benefits that may be necessary for induction of MRs but not for their later development, it may be worthwhile testing whether gelatinization and serum-coating can be omitted.

Note that MR2 and MR4 develop well in all cultured explants, but MR3 failed to develop in the majority of our culture experiments using this medium and metal support grids [5]. However, when we used BGJb medium (Sigma, St. Louis, MO) supplemented with 0.5 % penicillin/streptomycin and 0.2 % ascorbic acid in electroporation experiments as in [10], in combination with Millicell membrane inserts, MR3 always developed. We have not yet tested whether this higher success is due to the medium or the flat support of the membrane inserts as opposed to the quarter Track-Etch filters becoming concave during the culture, as their size is small compared to the hole in the metal grid. This BGJb medium with supplements is also used by [11] for bead implantation experiments. Although the authors of the latter publication do not mention success or failure rates of MR3 formation, this medium may be a good alternative to the one described in the current protocol. Prepare a 10 mg/ml stock solution of ascorbic acid (Cell culture tested, Sigma A4403) in distilled water, filter-sterilize (0.22 μm, Millipore), and store single-use aliquots at −20 °C.

Commercially available chick extract (several vendors) may be a practical alternative, but I have not tested these.

A movie showing embryo dissection (step 2) is available online at http://​www.​ijdb.​ehu.​es/​data/​11/​113424ls/​IntJDevBiol-113424ls-SuppMovie1.​mp4 [13], and needs to be downloaded (by opting “save link as…”) for viewing. Note that for this protocol, cleaning of surfaces with RNase-away is not necessary, and after dissection, embryos should NOT be collected in RNA-later, but in sterile DPBS.

A movie showing flank dissection (step 4) is available online at http://​www.​ijdb.​ehu.​es/​data/​11/​113424ls/​IntJDevBiol-113424ls-SuppMovie2.​mp4 [13], and needs to be downloaded (by opting “save link as …”) for viewing. Note that embryos in the movie were stored in RNA-later and thus look wrinkled/shriveled compared to the fresh embryos one uses for culture experiments. Once the visceral organs have been removed, the subsequent manipulations in this movie should NOT be followed for culture experiments.