Embryos were allowed to develop to early stage 8, and embryo extracts were prepared. To measure the level of ectopic lamin expression in microinjected embryos, a rabbit antibody against GFP was used at 1: For Experiment 4, HeLa cells were transiently transfected with plasmids expressing LA, LB1, or LB2 and co-transfected with an mCherry plasmid to allow for identification of transfected cells. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. A , Western blots were performed to estimate the endogenous amounts of different lamins present in X. A tribute to the Xenopus laevis oocyte and egg.
10 thoughts on “A Day in the Life of…a Xenopus lab”
Polarity in embryogenesis
One further preparation is required before my work can begin — the removal of the jelly coats that surround the embryos. In addition, in the s German embryologist Johannes Holtfreter was able to show that the dorsal marginal zone cells, i. Leave a Reply Cancel reply Your email address will not be published. It is well established that lamins are required for nuclear expansion during interphase, for example nuclei assemble but fail to grow in X. Add 5 ml of nuclear cushion buffer. In vivo cross-linking and immunoprecipitation for studying dynamic Protein: Aliquots from the same reaction were fixed and visualized by immunofluorescence using an antibody against the nuclear pore complex NPC mAb at:
Blower's present address is Dept. In keratinocytes lacking all lamins, NE and endoplasmic reticulum ER membranes spread into the chromatin In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. Critical steps within these protocols include: The process of gastrulation allows for the formation of the germ layers in metazoan embryos, and is generally achieved through a series of complex and coordinated cellular movements.
In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. An in vivo chemical library screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis and lymphangiogenesis. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. Do not excessively pipet the extract. Green indicates preparation of X.