Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of a nucleus into an embryo – egg cell – or...
Reexamination Certificate
1998-08-10
2001-12-18
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of a nucleus into an embryo, egg cell, or...
C800S014000, C800S008000
Reexamination Certificate
active
06331659
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the cloning of animals by the insertion of a nucleus of an adult somatic cell into an enucleated oocyte in such a way that the host oocyte forms an embryo and can develop into a live animal. In one embodiment of the invention, insertion of a nucleus is accomplished by piezo electrically-actuated microinjection.
The rapid production of large numbers of near-identical animals is very desirable. For example, it is expected that broad medical benefits may be obtained when the near-identical animals are also genetically engineered (e.g., transgenic) animals. Genetically altered large animals can act as living pharmaceutical “factories” by producing valuable pharmaceutical agents in their milk or other fluids or tissue (a production method sometimes referred to as “pharming”) or act as living organ or cell “factories” for human organs or cells that will not be rejected by the human immune system. The production of large numbers of near-identical research animals, such as mice, guinea pigs, rats, and hamsters is also desirable. For example, the mouse is a primary research model for the study of mammalian biology, and the availability of near-identical, transgenic or non-transgenic, mice would be very beneficial in the analysis of, for example, embryonic development, human diseases, and for testing of new pharmaceuticals. Thus, for a variety of reasons, (e.g., in the context of breeding farm animals, or the interpretation of data generated in mice), it may be desirable to reliably produce offspring of a particular animal that are genetically near-identical to the parent.
Further, with respect to transgenesis, current protocols for generating transgenic animals are not sufficiently advanced to guarantee the programmed control of gene expression in the context of the whole animal. Although it is possible to minimize detrimental “position” effects caused by the quasi-random manner in which the transgene integrates into the host genome, differences can exist in transgene expression levels between individuals carrying the same transgene construct inserted at the same locus in the same copy number. Thus, generating even modest numbers of transgenic animals producing the desired levels of any given recombinant protein(s) can be very time-consuming and expensive. These problems may be exacerbated because the number of transgenic offspring is often low (commonly only one) due to low efficiency, and many transgenic founders are infertile.
One approach to solving these problems is to “clone” genetically near-identical animals from the cells of transgenic or non-transgenic adult animals that have a desired trait or produce a target product at the desired level. To this end, colonies of genetically near-identical animals (clones) could be generated relatively rapidly from the cells of a single adult animal. Moreover, selective and reliable cloning of adult animals that produce increased yields of milk and meat could rapidly produce large numbers of high producers. Cloning of animals from adult somatic cells could also be beneficial in the reproduction of pets (e.g., dogs, cats, horses, birds, etc.) and rare or endangered species. As used herein, “cloning” refers to the full development to adulthood of an animal whose non-mitochondrial DNA may be derived from a somatic donor cell through the transfer of nuclear chromosomes from the somatic donor cell to a recipient cell (such as an oocyte) from which the resident chromosomes have been removed.
In normal mammalian development, oocytes become developmentally arrested at the germinal vesicle (GV) stage in prophase of the first meiotic division. Upon appropriate stimulation (e.g., a surge in plasma luteinizing hormone), meiosis resumes, the germinal vesicle breaks down, the first meiotic division is completed and the oocyte then becomes arrested at metaphase of the second meiosis (“Met II”). Met II oocytes can then be ovulated and fertilized. Once fertilized, the oocyte completes meiosis with the extrusion of the second polar body and the formation of male and female pronuclei. The embryos begin to develop by undergoing a series of mitotic divisions before differentiating into specific cells, resulting in the organization of tissues and organs. This developmental program ensures the successful transition from oocyte to offspring.
Although the cells of early embryos have classically been regarded as totipotent (that is, that they are capable of developing into a new individual per se), this totipotency is lost following a small number of divisions, that number varying between species (e.g., murine and bovine embryos). The mechanisms underlying this apparent loss of totipotency are poorly understood but are presumed to reflect subtle changes in the DNA environment affecting gene expression, that are collectively termed “reprogramming”. Without being bound by theory, it is believed that cloning techniques could possibly either subvert or mimic “reprogramming”.
Given the enormous practical benefits of cloning, there has been a commensurately great interest in overcoming technological barriers and developing new a techniques for the fusion of either embryonic cells or fetal cells with enucleated oocytes. To date, however, there has been a lack of reported protocols that have reproducibly generated full term development of clones from adult somatic cells. For example, it has been reported that when bovine cumulus cell nuclei were injected into enucleated oocytes which were then electro-activated, 9% of 351 injected oocytes developed to blastocysts, but none developed to term. Likewise, Sendai virus-mediated fusion of adult mouse thymocytes with enucleated Met II oocytes, followed by activation thirty to sixty minutes later with 7% ethanol, resulted in 75% of 20 oocytes reaching the 2-cell stage, but none developed beyond the 4-cell stage.
A recent report describes the electrofusion of cultured “mammary gland cells” with enucleated oocytes to produce a single live offspring sheep, which was named “Dolly” (Wilmut, I. et al. (1997),
Nature
385, 810-813). Dolly is reported to have developed from one of 434 enucleated oocytes electrofused with cells derived from the mammary gland that had been cultured for five days under conditions of serum starvation. According to the method reported to have been used to clone Dolly, the “mammary gland cell” was inserted by micropipette into the perivitelline space of an enucleated oocyte. Wilmut reports that the cells were immediately subjected to an electric pulse to induce membrane fusion and activate the oocyte to trigger resumption of the cell cycle. The resulting embryo (in addition to 28 others in the experiment) was transferred into a suitable recipient and, in this single case, the pregnancy proceeded to produce Dolly. However, because the “mammary gland cell” was from a cell line established from a 6-year old sheep that was in the third trimester of pregnancy, doubt has been publicly expressed as to the identity of the cells from which the donor nucleus was obtained, and even whether that cell was of adult origin.
In view of the foregoing, a controllable and efficient method of cloning animals from adult somatic cells is highly desirable. The present invention provides a novel method to achieve this.
SUMMARY OF THE INVENTION
The invention provides a method for cloning an animal from an adult somatic cell by directly inserting the nuclear contents of the somatic cell (or a portion of the nuclear contents including at least the minimum chromosomal material able to support development) into the cytoplasm of an enucleated oocyte, and facilitating embryonic development of the resulting cell to term and beyond. As used herein, the term “adult somatic cell” means a cell from a post-natal animal, which is therefore neither a fetal cell nor an embryonic cell, and which is not of the gamete lineage. The resulting viable offspring is a clone of the animal that originally provided the somatic cell nucleus for injection into the oocyte. The invention is applicable to cloning
Wakayama Teruhiko
Yanagimachi Ryuzo
Crouch Deborah
Jones Day Reavis & Pogue
University of Hawai'i
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