[Show abstract][Hide abstract] ABSTRACT: Transgenic mouse technology is a powerful method for studying gene function and creating animal models of human diseases. Currently, the most widely used method for generating transgenic mice is the pronuclear microinjection method. In this method, a transgenic DNA construct is physically microinjected into the pronucleus of a fertilized egg. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. A portion of the mice born to these surrogate mothers will harbor the injected foreign gene in their genomes. These procedures are technically challenging for most biomedical researchers. Inappropriate experimental procedures or suboptimal equipment setup can substantially reduce the efficiency of transgenic mouse production. In this chapter, we describe in detail our microinjection setup as well as our standard microinjection and oviduct transfer procedures.
[Show abstract][Hide abstract] ABSTRACT: Pronuclear microinjection is the most used method for generating transgenic mice. The quality of DNA to be microinjected is a key determinant of the success rate of this method. DNA purity is a critical factor because trace amounts of many substances, when microinjected into the pronucleus of the fertilized egg, can kill or prevent the further development of the embryo. Avoiding all contaminants is not a trivial issue, because most transgenic fragments need to be purified from agarose gels. Small particles and viscous materials in the DNA solution can also dramatically reduce the efficiency of microinjection because they tend to clog the injection needles. DNA shearing or breakage during purification and microinjection is also a potential problem, particularly when linearized bacterial artificial chromosomes (BAC) DNAs are used. The overall quantity and the final DNA concentration are also important considerations, because egg -pronuclei are very sensitive to the amount of foreign DNA. In this chapter, we first discuss the general guidelines and cautions for preparing microinjection-quality DNA, and then describe in detail two -protocols, one for gel purification of transgenic fragments from plasmid vectors and the other for isolating high-quality BAC DNA from bacteria.
[Show abstract][Hide abstract] ABSTRACT: As more and more genetically modified mouse lines are being generated, it becomes increasingly common to share animal models among different research institutions. Live mice are routinely transferred between animal facilities. Due to various issues concerning animal welfare, intellectual property rights, colony health status and biohazard, significant paperwork and coordination are required before any animal travel can take place. Shipping fresh or frozen preimplantation embryos, gametes, or reproductive organs can bypass some of the issues associated with live animal transfer, but it requires the receiving facilities to be able to perform delicate and sometimes intricate procedures such as embryo transfer, in vitro fertilization (IVF), or ovary transplantation. Here, we summarize the general requirements for live animal transport and review some of the assisted reproductive technologies (ART) that can be applied to shipping and reviving mouse lines. Intended users of these methods should consult their institution's responsible official to find out whether each specific method is legal or appropriate in their own animal facilities.
Methods in enzymology 12/2010; 476:37-52. DOI:10.1016/S0076-6879(10)76003-1 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.