Whole embryo culture (WEC) enables rodent embryos to be grown in vitro through the early stages of organogenesis, overcoming the inaccessibility of embryos within the uterus. WEC allows analysis of the direct effects of exogenous agents on the embryo, permits surgical/other interventions, and is used increasingly to study how gene dysfunction leads to birth defects. In terms of the 3Rs, WEC is an efficient way of avoiding the manipulation of pregnant female rodents. After Schedule 1 killing of the dam, littermate embryos can be allocated to different treatment groups, enhancing comparability between groups, and counteracting inter-litter variability. WEC is already widely used: e.g. 82 publications in 2010-14. However, several factors currently limit its more general uptake, and this PhD studentship aims to adapt WEC to overcome these limitations.
The dependence of WEC on lab-produced rat serum as culture medium deters some users. Moreover, commercial rat serum fails to support optimal embryo development. Recently, a serum-free, defined medium was reported for WEC, and our first aim is to assess its effectiveness compared with serum-based culture. A second limitation is the relatively poor molecular characterisation of embryos developing in culture. We will perform a direct transcriptomics comparison of embryos developing in WEC and in vivo, using RNAseq analysis. A third limitation is the lack of any protocol for inducing conditional transgene expression (e.g. ERT2Cre) in embryo culture. Our pilot study shows that tamoxifen induction works in WEC and this analysis will be extended into a workable method. Through these studies, the student will extend and validate WEC for contemporary birth defects research. He/she will then use the optimised techniques, particularly induction of conditional transgene expression, to perform a hypothesis-driven analysis of the role of the Vangl2 gene in development of mouse spina bifida.
Culshaw KH et al. (2019). Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium. Birth Defects Research 111(6):1-13. doi: 10.1002/bdr2.1538
Savery D et al. (2019). Refinement of inducible gene deletion in embryos of pregnant mice. Birth Defects Research 112(2):196-204. doi: 10.1002/bdr2.1628
Hughes A et al. (2018). Valproic acid disrupts the biomechanics of late spinal neural tube closure in mouse embryos. Mechanisms of Development 149:20-26. doi: 10.1016/j.mod.2017.12.001
Leung KY et al. (2017). Partitioning of One-Carbon Units in Folate and Methionine Metabolism Is Essential for Neural Tube Closure. Cell Reports 21(7):1795-1808. doi: 10.1016/j.celrep.2017.10.072