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
1998-01-08
2001-04-10
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, C800S015000, C800S016000, C800S017000, C435S325000
Reexamination Certificate
active
06215041
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cloning procedures in which cell nuclei derived from differentiated fetal or adult bovine cells, which include non-serum starved differentiated fetal or adult bovine cells, are transplanted into enucleated oocytes of the same species as the donor nuclei. The nuclei are reprogrammed to direct the development of cloned embryos, which can then be transferred to recipient females to produce fetuses and offspring, or used to produce cultured inner cell mass cells (CICM). The cloned embryos can also be combined with fertilized embryos to produce chimeric embryos, fetuses and/or offspring.
REFERENCES
The following publications, patent applications and patents are cited in this application as superscript numbers:
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22 Pedersen,
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23 Prather, et al.,
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24 Purcel, V. G. and Rexroad, Jr., C. E., Status of research with transgenic farm animals,
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25 Saito, et al.,
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All of the above publications, patent applications and patents are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Genetic modification of cattle could be useful in increasing the efficiency of meat and milk production. An ideal system for producing transgenic animals for agricultural applications would be highly efficient and use small numbers of recipient animals to produce transgenics. It would allow the insertion of a transgene into a specific genotype. The insertion would preferably be into a predetermined site which would confer high expression and not affect general viability and productivity of the animal. Furthermore, the identification of a locus for insertion would allow multiple lines to be produced and crossed to produce homozygotes and new genetic background could easily be added to the transgenic line by the production of new transgenics at any time. Therefore, the ideal system would likely require the transfection and selection of cells that could be easily grown in culture yet retain the potency to form germ cells and pass the gene to subsequent generations.
Various methods have been utilized in an attempt to genetically modify cattle so as to introduce superior agricultural qualities including pronuclear microinjection. One of the limitations of pronuclear microinjection is that the gene insertion site is random. This typically results in variations in expression levels and several transgeniclines must be produced to obtain one line with appropriate levels of expression to be useful. Because integration is random, it is advantageous that a line of transgenic animals be started from one founder animal, to avoid difficulties in monitoring zygosity and potential difficulties that might occur with interactions among multiple insertion sites.
8
Furthermore, starting a transgenic line from one hemizygous animal with a random insert would require breeding several generations and significant time for introgression of the transgene into the population before breeding and testing homozygotes if inbreeding is to be avoided.
8
Even without concern for inbreeding, it would take 6.5 years before reproduction could be tested in homozygous animals.
26
Finally, the quality of the genetics of a monozygous transgenic line would lag behind that of the general population because of the reduced population within which to select future generations of transgenic animals and the difficulty of bringing new genetics into a population in which the transgene is fixed.
A second limitation of the pronuclear microinjection procedure is its efficiency; which ranges from 0.34 to 2.63% of the gene-injected embryos developing into transgenic animals and a fraction of these appropriately expressing the gene.
24
This inefficiency results in a high cost of producing transgenic cattle because of the large number of recipients needed and, more importantly, unpredictability in the genetic background into which the gene is inserted because of the large number of embryos needed for microinjection. For agricultural purposes, a high quality genetic background is essential, therefore, long-term backcrossing strategies must be used with pronuclear microinjection. Thus, the ability to clone, or to make numerous identical genetic copies, of an animal comprising a desired genetic modification would be advantageous.
Another such system for producing transgenic animals has been developed and widely used in the mouse and involves the use of embryonic stem (ES) cells.
Embryonic stem cells in mice have enabled researchers to select for transgenic cells and perform gene targeting. This allows more genetic engineering than is possible with other transgenic techniques. Mouse ES cells are relatively easy to grow as colonies in vitro. The cells can be transfected by standard procedures and transgenic cells clonally selected by antibiotic resistance.
9
Furthermore, the efficiency of this process is such that sufficient transgenic colonies (hundreds to thousands) can be produced to allow a second selection for homologous recombinants.
9
M
Cibelli Jose
Golueke Paul
Jerry D. Joseph
Robl James M.
Stice Steven L.
Crouch Deborah
Teskin Robin L.
University of MMassachusetts
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