Abstract: A nuclear transfer method is provided wherein nuclear DNA in whole or part is injected into enucleated oocytes. The method is suitable for different donor cells, and preferably ES cells.

Abstract: The invention provides a method for generating transgenic animals and cells by the coinsertion of nucleic acid and a nucleus into an unfertilized oocyte. Preferably, the coinsertion is by microinjection and more preferably by piezo-electrically actuated microinjection. Transgene (tg) expressing embryos are here produced following coinjection of unfertilized mouse oocytes with sperm heads and exogenous DNA encoding either a green fluorescent protein (GFP) or &bgr;-galactosidase reporter. The microinjected oocyte may be allowed to develop into differentiated cells or stem cells; into an embryo in vitro prior to transfer into a host surrogate mother, or it may be transferred directly into a host surrogate mother. Embryonic development can occur to term, such that the offspring possess transgenic modifications that may alter their characteristics (phenotype) and are, in turn, transmitted to their offspring.

Abstract: A nuclear transfer method is provided wherein nuclear DNA in whole or part is injected into enucleated oocytes. The method is suitable for different donor cells, and preferably ES cells.

Abstract: The invention provides a microinsertion-based, trans-complementation method of in vitro oocyte activation useful to identify properties of sperm-borne oocyte-activating factor(s) (SOAF) and ooplasmic interactions with sperm components at fertilization. The invention provides at least one detergent-insoluble heat-sensitive component of the perinuclear matrix of a spermatozoon (SOAFm) that acts coordinately with at least one heat-stable submembrane sperm component. SOAFm is solubilized in vitro (SOAFs) under reducing conditions similar to those encountered in the ooplasm. By the method of the invention, the failure of heat-inactivated demembranated sperm heads to activate an egg is rescued by coinsertion of SOAFs into an oocyte. SOAFs is protease-sensitive and is liberated from demembranated heads in a temperature-dependent manner that inversely correlates with the ability of demembranated sperm heads to activate oocytes.