Pronuclear microinjection

Fig. 1. Outline of the generation of transgenic mice via pronuclear microinjection. Transgenic animals carry the transgenes randomly integrated (hemizygous, tandems, dominant).

Although technically simple, the use of retroviruses and retroviral vectors has not found widespread application. This is mainly due to the size limitations for transfected DNA (about 10 kb), as well as to the unresolved problems of reproducibly expressing a virally transfected eukaryotic gene. However, the methods of pronuclear microinjection and the use of embryonic stem cells are being successfully applied by a growing number of research groups. 

Wall, R. J. (2001) Pronuclear microinjection. Cloning Stem Cells 3, 209-220. 

Schedl, A., Larin, Z., Montoliu, L., Thies, E., Kelsey, G., Lehrach, H., and Schutz, G. (1993) A method for the generation ofYAC transgenic mice by pronuclear microinjection. Nucl. Acids Bes. 21, 4783 1787. 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

---