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(12) United States Patent (10) Patent No.: US 7,199,281 B2 Murray Et Al US007 199281B2 (12) United States Patent (10) Patent No.: US 7,199,281 B2 Murray et al. (45) Date of Patent: Apr. 3, 2007 (54) METHOD OF GENERATING ATRANSGENIC Seamark, 1994, Reproductive Fertility and Development, 6:653-7.* LIVESTOCK ANIMAL Mullins, 1996, J. Clin. Invest., vol. 98, pp. S37-S40.* McCreath, 2000, Nature, vol. 405, pp. 1066-1069.* (75) Inventors: James D. Murray, Davis, CA (US); Denning, C. 2001 A. Nature Biotechnology, vol. 19, pp. 559-562.* Elizabeth A. Maga, Sacramento, CA Dinnyes, 2002, Cloning and Stem Cells, vol. 4, pp. 81-90.* (US); Gary B. Anderson, Davis, CA Denning, C., 2001B, Gene Targeting from primaty fetal fibroblasts (US); Stefanie M. Oppenheim, Davis, from sheep and pig, Cloning and Stem Cells, 3:221-231.* CA (US) Poljaeva, I.A., 2000, Nature, 407:86-90.* Clark, A.J., 2000, Gene targeting in livestock: a preview, Transgenic (73) Assignee: The Regents of the University of Research, 9:263-275.* California, Oakland, CA (US) Capecchi, 1994, Targeted Gene Replacement, Scientific American, vol. 270, pp. 34-41.* (*) Notice: Subject to any disclaimer, the term of this http://en.wikipedia.org/wiki/Livestock.* patent is extended or adjusted under 35 Harrison, SJ et al., 2002, Efficient generation of 1(1.3) U.S.C. 154(b) by 412 days. galactosyltransferase knockout porcine fetal fibroblasts for nuclear transfer, Transgenic Research, 11:143-150.* (21) Appl. No.: 10/238,042 Poljaeva I.A. and Campbell, KHS, 2000, New advances in somatic cell nuclear transfer:application in transgenesis, Theriogenology, (22) Filed: Sep. 6, 2002 53:117-126. Thomson, AJ et al., 2003, Gene targeting in livestock, Reproduction (65) Prior Publication Data Supplement, 61:495-508.* US 2003/O115618 A1 Jun. 19, 2003 Akhmedov et al. “Characterization of two nuclear mammalian homologous DNA-pairing activities that do not require associated Related U.S. Application Data exonuclease activity” Proc. Natl. Acad Sci. USA vol. 92, pp. 1729 1733, Feb. 1995. (60) Provisional application No. 60/317,915, filed on Sep. Liu et al., “Insulin-Like Growth Factor-I Affects Perinatal Lethality 7, 2001. and Postnatal Development in a Gene Dosage-Dependent Manner: Manipulation Using the CrefloxP System In Transgenic Mice” (51) Int. Cl. Molecular Endocrinology (1998), vol. 12, No. 9, 1452-1462. CI2N IS/00 (2006.01) Plug et al., “Presynaptic associate of RadS1 protein with selected AIK 67/027 (2006.01) sites in meiotic chromatin” Proc. Natl. Acad. Sci. USA vol. 93, pp. (52) U.S. Cl. ............................ 800/25: 800/14: 800/15; 5920-5924, Jun. 1996. 800/16: 800/17 Utomo, A.R.H. “Temporal, Spatial, and Cell Type Specific Control (58) Field of Classification Search ..................... None of Cre-Mediated DNA Recombination in Transgenic mice” Nature See application file for complete search history. Biotechnology, Nov. 1999, vol. 17, 1091-1098. (56) References Cited * cited by examiner U.S. PATENT DOCUMENTS Primary Examiner Valarie Bertoglio (74) Attorney, Agent, or Firm Paula A. Borden; Bret Field; 5,763,240 A 6/1998 Zarling et al. Bozicevic, Field & Francis 5.948,653 A 9, 1999 Pati et al. 6,074,853. A 6, 2000 Pati et al. (57) ABSTRACT 6,200,812 B1 3, 2001 Pati et al. 6.255,113 B1 7/2001 Zarling et al. FOREIGN PATENT DOCUMENTS The present invention provides methods of producing trans genic livestock animals. The methods generally involve first WO WO 99.6O108 11, 1999 introducing a nucleoprotein made up of nucleic acid and a recombinase into a totipotent or pluripotent cell to produce OTHER PUBLICATIONS a recombinant totipotent or pluripotent cell and then grow Maga, E. The use of recombinase proteins to generate transgenic ing the recombinant totipotent or pluripotent cell to produce large animals, Cloning and Stem Cells, 3:233-241, 2001.* the transgenic livestock animal. The invention further pro Niemann, 1997, Transg. Res. vol. 7, pp. 73-75.* vides kits for use in generating transgenic non-human ani Kilby, 1993, Trends in Genetics, 9:413-421.* mals of the invention. Moreadith, 1997, Gene targeting in embryonic stem cells: the new physiology and metabolism, Journal of Molecular Medicine, vol. 75, pp. 208-216.* 8 Claims, 3 Drawing Sheets U.S. Patent Apr. 3, 2007 Sheet 1 of 3 US 7,199,281 B2 1 2 3 4. 12.3.4 - +RecA --RecA -D - RecA Figure 1A Figure 1B U.S. Patent Apr. 3, 2007 Sheet 2 of 3 US 7,199,281 B2 Figure 2A. Figure 2B. M 1 2 3 4 5 6 7 o C M 2 3 4 5 6 7 8 9 O () - - M s: Figure 2C. 1 2 3 4 5 6 7 8 Figure 2D. Mu e u e u eu e u e Li eu e u e 9 - mm M M 2 3 4 5 6 7 8 9 - -- US 7,199,281 B2 1. 2 METHOD OF GENERATING ATRANSGENIC or by cloning, both of which allow for the targeted insertion LIVESTOCK ANIMAL of DNA into cells in culture. The important feature of these methods for the production of transgenic animals is that both CROSS-REFERENCE TO RELATED ES cells or any donor cell (i.e., the differentiated somatic APPLICATIONS cell) to be used in nuclear transfer can be grown in culture and genetically modified with a desired transgene. The Pursuant to 35 U.S.C. S 119 (e), this application claims modified cells can then be selected, characterized prior to priority to the filing date of the U.S. Provisional Patent being used to generate transgenic animals. The potential Application Ser. No.: (a) 60/317,915 filed Sep. 7, 2001; the advantages these methods offer over pronuclear microinjec disclosure of which are herein incorporated by reference. 10 tion include the ability to do gene targeting, thereby allow ing for the creation of knockouts and enabling the modifi INTRODUCTION cation of endogenous genes. Also, with cloning, all animals born will be germ line transgenic. However, identifying the 1. Field of the Invention homologous recombinants in a large population of non The field of this invention is transgenic non-human ani 15 homologous random integrants often proves to be the rate mals. limiting step for creating homologously modified mamma 2. Background of the Invention lian cell lines. This severely limits the ability to manipulate The overall goal in making a transgenic animal is the target genes systematically. stable introduction of a desired DNA sequence into the germ These strategies are labor-intensive, time-consuming, and line of the host animal that can be transmitted to offspring in ultimately limit homologous recombination genetic engi a Mendelian fashion. By incorporating new or modified neering of mammalian cells for commercial applications. genes at the genetic level, the characteristics of the animal Other disadvantages include the fact that currently, among can be specifically changed. Transgenic animals are gener mammals, ES cells are available only for mice. While ated for a variety of purposes. They can be used as basic nuclear transfer allows for targeted modifications in live research models, specialized non-agricultural purposes 25 stock species, it is not Supportive with all cell types, requires (such as pharmaceutical production or Xenotransplantation) specialized techniques and conditions, is hard to maintain and also to enhance animal production traits and products. pregnancies and is associated with large offspring syndrome. For many applications, large animals, e.g., livestock Such as Moreover, the process is also very inefficient. The efficiency pigs, cows, sheep, and goats, are of interest. Producing and frequency with which transgenic animals are generated transgenic livestock is not as efficient as mice and is an 30 with these methods are in the same range as those of the expensive and time-consuming process. Accordingly, there more established and simpler method of pronuclear micro is much interest in developing methods that increase the injection. efficiency and specificity of the transgenic process in non Presently, nuclear transfer efficiency in sheep is around murine large animals. 0.04–1.7% live born animals from reconstructed embryos, Transgenic animals are generally produced by one of 35 which is similar to standard pronuclear microinjection trans three main methods: 1) the pronuclear microinjection of genic rates of approximately 1%. fertilized one-cell embryos followed by reimplantation into Thus, there is a need in the art for methods of increasing Surrogate mothers; 2) the genetic manipulation of embryonic the efficiency of generating transgenic animals, particularly stem (ES) cells followed by introduction of modified ES livestock. The present invention addresses this need. cells into developing embryos; and 3) by the genetic 40 manipulation of Somatic primary cells followed by nuclear Literature transfer into a recipient oocyte. The standard and most U.S. Pat. Nos. 5,763,240; 5,948,653; 6,074.853; 6,200, established method of producing transgenic animals such as 812: 6,255,113. mice, rabbits, pigs, goats, or cows generally rely on the SUMMARY OF THE INVENTION microinjection of DNA encoding a transgene into the pro 45 nucleus of fertilized Zygotes. However, this method cur The present invention provides methods of producing rently has several unavoidable shortcomings. transgenic livestock animals. The methods generally involve Pronuclear microinjection methods generally result in the first introducing a nucleoprotein made up of nucleic acid and random integration of transgenes in the chromosome of the a recombinase into a totipotent or pluripotent cell to produce Zygote. If the DNA construct is integrated into an inactive 50 a recombinant totipotent or pluripotent cell and then grow region of chromosome, it is unlikely to be expressed. As a ing the recombinant totipotent or pluripotent cell to produce consequence, it is necessary to generate several founders and the transgenic livestock animal. The invention further pro carry out extensive characterizations on them all in order to vides kits for use in generating transgenic non-human ani identify a line of animals that will stably express the mals of the invention.
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