Abstract: The present invention relates to methods for obtaining transgenic embryos, animals, and plants. Also encompassed in the present invention are transposase-mediated trangenesis methods including transposase-mediated intracytoplasmic sperm injection (TN:ICSI), transposase-mediated intracytoplasmic round spermatid injection (TN:ROSI), and transposase-mediated in vitro fertilization (TN:IVF).

Abstract: The present invention provides a composition for freezing or freeze-drying spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads thereof to maintain chromosome integrity at a temperature of about +4° C. to about ?200° C. Also provided is a container containing the composition. The present invention further provides a method for maintaining chromosome integrity of spermatozoa or sperm heads during freezing or freeze-drying. The present invention also provides methods for freezing or freeze-drying spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring. Also provided are frozen or freeze-dried spermatozoa produced by these methods, and containers containing the frozen or freeze-dried spermatozoa. Finally, the present invention provides methods for producing a live mammalian offspring from an oocyte fertilized with a rehydrated freeze-dried (or thawed or freeze-thawed) spermatozoan.

Abstract: The invention provides methods for producing transgenic embryos and animals with a higher efficiency than presently available procedures based on Intracytoplasmic Sperm Injection (ICSI) of nucleic acid complexed with recombinase.

Abstract: The present invention provides methods of preparing spermatozoa suitable for use in ICSI-mediated transgenesis, wherein the methods include the suspension of spermatozoa in a buffered medium comprising an ion-chelating agent. In a preferred embodiment of the invention, the method of preparing spermatozoa for ICSI-mediated transgenesis further comprises treatment of membrane-disrupted or demembranated spermatozoa with a disulfide reducing agent. Also provided are spermatozoa suitable for use in ICSI-mediated transgenesis, wherein the exogenous nucleic acid to be co-inserted in an unfertilized oocyte via ICSI is closely associated with the membrane-disrupted or demembranated spermatozoon.

Abstract: A method of producing a non-human mammalian embryo, such as a mouse embryo, by nuclear cloning, in which the nucleus from a non-human mammalian embryonic stem (ES) cell (e.g., a non-human mammalian F1 ES cell), such as the nucleus of a mouse F1 ES cell, is introduced into an enucleated non-human mammalian oocyte, such as an enucleated mouse oocyte; embryos produced by the method; a method of producing mice from the resulting embryos and the mice produced thereby.

Abstract: The present invention provides a composition for freezing or freeze-drying spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads thereof to maintain chromosome integrity at a temperature of about +4° C. to about −200° C. Also provided is a container containing the composition. The present invention further provides a method for maintaining chromosome integrity of spermatozoa or sperm heads during freezing or freeze-drying. The present invention also provides methods for freezing or freeze-drying spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring. Also provided are frozen or freeze-dried spermatozoa produced by these methods, and containers containing the frozen or freeze-dried spermatozoa. Finally, the present invention provides methods for producing a live mammalian offspring from an oocyte fertilized with a rehydrated freeze-dried (or thawed or freeze-thawed) spermatozoan.

Abstract: The invention provides a method for freeze-drying spermatozoa to obtain at least one reconstituted spermatozoon whose head (nucleus) is capable of fertilizing an oocyte to produce a live offspring. The motility of spermatozoa which have been freeze-dried and stored in a vacuum at room temperature is not restored when rehydrated. Their plasma membranes are disrupted and they are all “dead” in the conventional sense. However, when they are injected microsurgically into oocytes, their nuclei transform into male pronuclei and participate in normal embryonic development.

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.

Abstract: Coinjection of unfertilized mouse oocytes with sperm heads and exogenous nucleic acid encoding a transgene results in transgene-expressing embryos, reflecting nucleic acid-sperm head association before coinjection. Nonselective transfer to surrogate mothers of embryos resulting from coinjection produced offspring expressing the integrated transgene.

Abstract: Animals are produced following injection of adult cumulus or fibroblast cell nuclei into enucleated oocytes. The invention provides a method for cloning an animal by directly inserting an adult somatic cell nucleus into a recipient enucleated oocyte. Preferably, the nucleus is inserted by microinjection and, more preferably, by piezo electrically-actuated microinjection. The oocyte is activated prior to, during, or up to about 6 hours after insertion of the nucleus, by electroactivation or exposure to a chemical activating agent, such as Sr2+. The activated renucleated oocyte is allowed to develop into an embryo and is transplanted to a host surrogate mother to develop into a live offspring.

Abstract: Animals are produced following injection of adult somatic cell nuclei into enucleated oocytes. The invention provides a method for cloning an animal by directly inserting at least a portion of the adult somatic nucleus (including the minimum chromosomal material able to support development) into a recipient enucleated oocyte. Preferably, the nucleus is inserted by microinjection and, more preferably, by piezo electrically-actuated microinjection. The oocyte is activated prior to, during, or up to about 6 hours after insertion of the nucleus, by electroactivation or exposure to a chemical activating agent, such as Sr2+. The activated renucleated oocyte is allowed to develop into an embryo and is transplanted to a host surrogate mother to develop into a live offspring.

Abstract: The invention provides a method for obtaining a live offspring having maternal chromosomes derived from a live first polar body of an oocyte that has completed the first meiotic division. It has been discovered herein that chromosomes in the first polar body are able to participate in normal embryonic development if they are allowed to complete the second meiotic division within an enucleated mature oocyte, and are then allowed to mingle with chromosomes of a spermatozoon. The invention further provides a method for producing up to four embyros or live offspring having the chromosomes of a single oocyte, by using both the first polar body chromosomes, and the second polar body chromosomes to reconstitute recipient enucleated (fertilized) oocytes.