# US 6,297,029 B1 · Mammalian artificial chromosomes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,297,029 B1 - Document ID: US6297029B1 - Status: Granted - Kind: B1 - Application number: 09/254,028 - Filed: 1999-09-28 - Granted: 2001-10-02 - Earliest claimed: 1999-09-28 - Inventors: Tsuneko Okazaki; Hiroshi Masumoto; Masashi Ikeno; Howard J. Cooke; Brenda Rose Grimes - Web page: https://patentgrep.com/patent/US6297029B1 ## CPC - C12N 15/85: for animal cells - C12N 15/81: for yeasts - A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy - C12N 2800/206: of yeast origin, e.g. YAC, 2u - C12N 2800/208: of mammalian origin, e.g. minichromosome - C12N 2840/20: translation of more than one cistron - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract A mammalian chromosome is produced according to the method comprising the steps of: introducing a DNA construct comprising a mammalian telomere and a centromere into a mammalian cell, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence consisting of: 5′-NTTCGNNNNANNCGGGN-3′, wherein N is any one of A, T, C and G. ## Claims ### Claim 1 A DNA construct comprising: a mammalian telomere and a DNA sequence of a mammalian centromere, wherein the DNA construct forms an artificial chromosome when introduced into mammalian cells, autonomously replicates in mammalian cells, is maintained extra chromosomally and is transmitted to progeny cells thereof, and wherein said DNA sequence contains a plurality of copies of the sequence: 5′-NTTCGNNNNANNCGGGN-3′, wherein N is selected from the group consisting of A, T, C and G. ### Claim 2 A recombinant DNA construct comprising: a mammalian telomere, a mammalian centromere comprising tandem repeats of the sequence: 5′-NTTCGNNNNANNCGGGN-3′, wherein N is any one of A,T,C and G and wherein the sequence is provided in a quantity that causes the DNA construct to form a mammalian artificial chromosome when introduced into a mammalian cell. ### Claim 3 (depends on 2) The recombinant DNA construct of claim 2, further comprising a sequence encoding a selectable marker gene. ### Claim 4 (depends on 2) The recombinant DNA construct of claim 2, which further comprises a genome DNA sequence containing a structural region and its regulatory region. ### Claim 5 (depends on 2) The recombinant DNA construct of claim 2, wherein said mammalian cell is a mouse cell. ### Claim 6 (depends on 2) The recombinant DNA construct of claim 2, wherein said mammalian cell is a human cell. ### Claim 7 (depends on 2) The recombinant DNA construct of claim 2, further comprising at least one DNA sequences that permits said DNA construct to multiply in yeast cells. ### Claim 8 A recombinant DNA construct comprising: a mammalian telomere, a mammalian centromere comprising tandem repeats of the sequence designated as Seq. No. 2, and wherein the sequence is provided in a quantity that causes the DNA construct to form a mammalian artificial chromosome when introduced into a mammalian cell. ### Claim 9 (depends on 8) The recombinant DNA construct of claim 8, further comprising a sequence encoding a selectable marker gene. ### Claim 10 (depends on 8) The recombinant DNA construct of claim 8, which further comprises a genome DNA sequence containing a structural region and its regulatory region. ### Claim 11 (depends on 8) The recombinant DNA construct of claim 8, wherein said mammalian cell is a mouse cell. ### Claim 12 (depends on 8) The recombinant DNA construct of claim 8, wherein said mammalian cell is a human cell. ### Claim 13 (depends on 8) The recombinant DNA construct of claim 8, further comprising at least one DNA sequences that permits said DNA construct to multiply in yeast cells. ### Claim 14 A recombinant DNA construct comprising: a mammalian telomere, mammalian centromere comprising a sequence that is capable of hybridizing under selective conditions with a sequence selected from the group consisting of Seq. No. 2, Seq. No. 3, Seq. No. 4, Seq. No. 5, Seq. No. 6 and Seq. No. 7. ### Claim 15 (depends on 14) The recombinant DNA construct of claim 14, wherein said sequence is a cloned human alphoid DNA of about 100 kb and is capable of hybridizing under selective conditions with Seq. No. 2. ### Claim 16 (depends on 14) The recombinant DNA construct of claim 14, further comprising a sequence encoding a selectable marker gene. ### Claim 17 (depends on 14) The recombinant DNA construct of claim 14, which further comprises a genome DNA sequence containing a structural region and its regulatory region. ### Claim 18 (depends on 14) The recombinant DNA construct of claim 14, wherein said mammalian cell is a mouse cell. ### Claim 19 (depends on 14) The recombinant DNA construct of claim 14, wherein said mammalian cell is a human cell. ### Claim 20 (depends on 14) The recombinant DNA construct of claim 14, further comprising at least one DNA sequence that permits said DNA construct to multiply in yeast cells. ### Claim 21 A cell line deposited at the Agency of Industrial Science and Technology, National Institute of Bioscience and Human Technology and designated as FERM BP-5625. ### Claim 22 (depends on 21) A DNA sequence comprising alfa 7C5 h TEL isolated from the cell line of claim 21. ### Claim 23 (depends on 2) A mammalian cell having a mammalian artificial chromosome that comprises the recombinant DNA construct of claim 2. ### Claim 24 (depends on 23) The mammalian cell of claim 23, wherein said mammalian cell is a mouse cell. ### Claim 25 (depends on 23) The mammalian cell of claim 23, wherein said mammalian cell is a human cell. ### Claim 26 (depends on 8) A mammalian cell having a mammalian artificial chromosome that comprises the recombinant DNA construct of claim 8. ### Claim 27 (depends on 26) The mammalian cell of claim 26, wherein said mammalian cell is a mouse cell. ### Claim 28 (depends on 26) The mammalian cell of claim 26, wherein said mammalian cell is a human cell. ### Claim 29 (depends on 14) A mammalian cell having a mammalian artificial chromosome that comprises the recombinant DNA construct of claim 14. ### Claim 30 (depends on 29) The mammalian cell of claim 29, wherein said mammalian cell is a mouse cell. ### Claim 31 (depends on 29) The mammalian cell of claim 29, wherein said mammalian cell is a human cell. ### Claim 32 (depends on 2) A method of making a mammalian artificial chromosome comprising: introducing the DNA construct of claim 2 into a mammalian cell and permitting the DNA construct to form a mammalian artificial chromosome within the mammalian cell. ### Claim 33 (depends on 8) A method of making a mammalian artificial chromosome comprising: introducing the DNA construct of claim 8 into a mammalian cell and permitting the DNA construct to form a mammalian artificial chromosome within the mammalian cell. ### Claim 34 (depends on 14) A method of making a mammalian artificial chromosome comprising: introducing the DNA construct of claim 14 into a mammalian cell and permitting the DNA construct to form a mammalian artificial chromosome within the mammalian cell. ### Claim 35 (depends on 1) A method of making an exogenous mammalian gene product, comprising: introducing the DNA construct of claim 1 into a mammalian cell, wherein the DNA construct of claim 1 further comprises an exogenous DNA sequence encoding the mammalian gene product and expressing the exogenous mammalian gene product. ### Claim 36 (depends on 2) A method of making an exogenous mammalian gene product, comprising: introducing the DNA construct of claim 2 into a mammalian cell, wherein the DNA construct of claim 2 further comprises an exogenous DNA sequence encoding the mammalian gene product and expressing the exogenous mammalian gene product. ### Claim 37 (depends on 8) A method of making an exogenous mammalian gene product, comprising: introducing the DNA construct of claim 8 into a mammalian cell, wherein the DNA construct of claim 8 further comprises an exogenous DNA sequence encoding the mammalian gene product and expressing the exogenous mammalian gene product. ### Claim 38 (depends on 1) The DNA construct of claim 1, wherein said DNA sequence is Seq. No. 2. ### Claim 39 (depends on 1) The DNA construct of claim 1, wherein said DNA sequence is Seq. No. 3. ### Claim 40 (depends on 1) The DNA construct of claim 1, further comprising: a yeast telomere, a yeast centromere and a yeast replication origin, wherein said DNA construct also forms an artificial chromosome when introduced into yeast cells, autonomously replicates in yeast cells, is maintained extra chromosomally and is transmitted to progeny cells thereof. ### Claim 41 (depends on 1) The DNA construct of claim 1, wherein said DNA sequence is derived from human chromosome 21. ### Claim 42 (depends on 1) The DNA construct of claim 1, further comprising a sequence encoding a selectable marker gene. ### Claim 43 (depends on 1) The DNA construct of claim 1, which further comprises a genome DNA sequence containing a structural region and its regulatory region. ### Claim 44 (depends on 1) The DNA construct of claim 1, wherein said mammalian cell is a mouse cell. ### Claim 45 (depends on 1) The DNA construct of claim 1, wherein said mammalian cell is a human cell. ### Claim 46 (depends on 1) The DNA construct of claim 1, further comprising at least one DNA sequence that permits the DNA construct to multiply in yeast cells. ### Claim 47 (depends on 1) A mammalian cell comprising the DNA construct of claim 1. ### Claim 48 (depends on 1) A yeast cell comprising the DNA construct of claim 1. ### Claim 49 A method of performing homologous recombination comprising: transfecting a recombinant DNA construct into a host cell that does not possess the gene for DNA recombination within its DNA, wherein the recombinant DNA construct may be carried during mitotic division of the host cell within a plasmid having a gene for DNA recombination, which plasmid is capable of being eliminated during mitotic division of the host cell, and collecting host cells that possess the recombinant DNA construct, but do not possess the plasmid. ### Claim 50 (depends on 1, 49) The method of claim 49, wherein said recombinant DNA construct is the DNA construct of claim 1. ### Claim 51 (depends on 2, 49) The method of claim 49, wherein said recombinant DNA construct is the DNA construct of claim 2. ### Claim 52 (depends on 8, 49) The method of claim 49, wherein said recombinant DNA construct is the DNA construct of claim 8. ### Claim 53 (depends on 14, 49) The method of claim 49, wherein said recombinant DNA construct is the DNA construct of claim 14. ### Claim 54 (depends on 49) The method of claim 49, wherein said host cell is a yeast cell.