USOO8703482B2 (12) United States Patent (10) Patent No.: US 8,703,482 B2 Oshimura et al. (45) Date of Patent: Apr. 22, 2014 (54) HUMAN ARTIFICIAL CHROMOSOME (HAC) Rasheed et al. “Characterization of a Newly Derived Human Sar VECTOR coma Cell Line (HT-1080).” Cancer, 1974, vol. 33, pp. 1027-1033. R. Moreadith etal, “Gene targeting in embryonic stem cells: the new physiology and metabolism.” J Mol Med, 1997, vol. 75, pp. 208-216. (75) Inventors: Mitsuo Oshimura, Tottori (JP); L. Mullins et al., “Perspective Series: Molecular Medicine in Geneti Motonobu Katoh, Tottori (JP); Kazuma cally Engineered Animals: Transgenesis in the Rat and Larger Ani Tomizuka, Gunma (JP); Yoshimi mals,” Journal of Clinical Investigation, 1996, vol. 97, No. 7, pp. Kuroiwa, Gunma (JP); Minoru 1557-1560. Kakeda, Gunma (JP) M. Pera et al. “Human embryonic stem cells,” Journal of Cell Sci ence, 2000, vol. 113, pp. 5-10. 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EP 1106061 A1 6, 2001 (Continued) WO 02/070648 A2 9, 2002 Primary Examiner — Kevin Hill OTHER PUBLICATIONS (74) Attorney, Agent, or Firm — Sughrue Mion, PLLC Korean Office Action dated Oct. 18, 2010, as issued in Korean Patent (57) ABSTRACT Application No. 10-2005-7005701. The present invention relates to a human artificial chromo Kuroiwa, Yoshimi et al., “Manipulation of human minichromosomes some (HAC) vector and a method for producing the same. The to carry greater than megabase-sized chromosome inserts'. Nature present invention further relates to a method for introducing Biotechnology, vol. 18, pp. 1086-1090, Oct. 18, 2000. foreign DNA using a human artificial chromosome vector and Grimes, Brenda R., et al., “Alpha-Satellite DNA and Vector Compo a method for producing a cell which expresses foreign DNA. sition Influence Rates of Human Artificial Chromosome Formation', Furthermore, the present invention relates to a method for Molecular Therapy, vol. 5, No. 6, pp. 798-805, May 2002. producing a protein. T. Shinohara et al. "Stability of transferred human chromosome fragments in cultured cells and in mice.” Chromosome Research, 5 Claims, 22 Drawing Sheets 2000, vol. 8, pp. 713-725. (12 of 22 Drawing Sheet(s) Filed in Color) US 8,703.482 B2 Page 2 (56) References Cited H. Masumoto et al., “Assay of centromere function using a human OTHER PUBLICATIONS artificial chromosome" 1998, Chromosoma vol. 107, pp. 406-416. European Search Report dated Feb. 15, 2006 for counterpart appli- Japanese Office Action issued Sep. 20, 2011 for counterpart Japanese cation PCT/JP0312734 to parent U.S. Appl. No. 10/530,207. application 2009-185530. U.S. Patent Apr. 22, 2014 Sheet 1 of 22 US 8,703.482 B2 targeting vecter K K: Kpn wild-type K K -X-----X---X-1a-------X. Xx 8X -l -ms-- w8XX&&888&SXX Cer probeS&8:8 te 9. Okto-orbi targeted allele K K U.S. Patent Apr. 22, 2014 Sheet 2 of 22 US 8,703.482 B2 2ip13 Drug resistant clones O e Polymorphism and o - name of gene markers or or cro - PRED65 | PRED3 21g2. - D21S265 CBR D21 S268 222.3 D2S259 Chronosome 2 U.S. Patent Apr. 22, 2014 Sheet 3 of 22 US 8,703.482 B2 Fig. 3 es S.e as o Drug resistant clones gig O C. c. 83 r kb 13.6 m. Honologous recombinant allele 9. Ouw wild-type allele U.S. Patent Apr. 22, 2014 Sheet 4 of 22 US 8,703.482 B2 Fig. 4 U.S. Patent Apr. 22, 2014 Sheet 5 of 22 US 8,703,482 B2 targeting vecter A. pCMVbsr loxP 3'-neo B -ana-(-a-ana & wild-type allele." r a' s 0. X " " ' " . 8.5 kb- : Xbal site homogous recombination in DT40 targeted allele A pcMvbsr loxP 3'-neo B U.S. Patent Apr. 22, 2014 Sheet 6 of 22 US 8,703.482 B2 S. Blasticidin resistant DT40 cones 35s& 3 & 8 is R s to kb 8.5 or m wild-type allele 7.6 “r rhomologous recombinant allele proximat region PCR amplification on the site generated by homologous distal recontration region U.S. Patent Apr. 22, 2014 Sheet 7 of 22 US 8,703.482 B2 Fig. 7 U.S. Patent Apr. 22, 2014 Sheet 8 of 22 US 8,703.482 B2 Fig. 8 Plasmid Peay GFP N \ E: EcoRI / BBan Pcy u/ oxP wild-type E. E. oxP 3. 3 mXXXXXX&X-X mid- --&-X-X-X {mb-mid PCMvBsd "... neo Homologous recombination targeted allele , E . B W-WXW-W--WWSWSWWWL-mid-4-(-m) -->{-> --- Pchy Es PolyEGFP Poly neo U.S. Patent Apr. 22, 2014 Sheet 9 of 22 US 8,703.482 B2 U.S. Patent Apr. 22, 2014 Sheet 10 of 22 US 8,703.482 B2 1 2 3 4 56 78 thresk res, U.S. Patent Apr. 22, 2014 Sheet 11 of 22 US 8,703.482 B2 Fig.11 targeting vecter H: find III te --mm- C targeted allele ygro H 5.8k hyg968 --> 5.9 kb - #21 p96705 N N -------- N Nsi I 1.4 kb 2.6 ko 1.9 kb U.S. Patent Apr. 22, 2014 Sheet 12 of 22 US 8,703.482 B2 Fig. 12 Polymorphism and names of gene markers 21g2. 2c22.11 2 a.22. 12 21q22.13 2 (22.2 Chronosome 21 U.S. Patent Apr. 22, 2014 Sheet 13 of 22 US 8,703.482 B2 Fig. 13 as s Hygromycin resistant s s DT40 clones . R ; U.S. Patent Apr. 22, 2014 Sheet 14 of 22 US 8,703.482 B2 Fig. 14 Hygromycin resistant e DAO cores s. g . 5.9 on xx PCR amplification by s Hyggé8/#21 p98705 Digestion of amplified product with Nis U.S. Patent Apr. 22, 2014 Sheet 15 of 22 US 8,703.482 B2 Fig. 15 U.S. Patent Apr. 22, 2014 Sheet 16 of 22 US 8,703,482 B2 U.S. Patent Apr. 22, 2014 Sheet 17 of 22 US 8,703.482 B2 Fig. 17 U.S. Patent Apr. 22, 2014 Sheet 18 of 22 US 8,703.482 B2 Fig. 18 U.S. Patent Apr. 22, 2014 Sheet 19 of 22 US 8,703.482 B2 -- - - - - - -- -------- -- - - - - -- ++++++ | + + + + + + + + + + + -- -- -- -- I -- 6|-61-I -- - - - -- + + + -- -- - - - - - - - - - -- -- - - -- -- - - - - - - - - - - - - - -- U.S.