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Chromosome-based platforms

a technology of chromosomes and platforms, applied in the field of chromosome-based platforms, can solve the problems of time-consuming and tedious processes

Inactive Publication Date: 2006-11-02
GLAXO GRP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Facilitates rapid and efficient integration of heterologous nucleic acids into artificial chromosomes, enabling tractable engineering and stable expression in mammalian and plant cells, with applications in transgenic animals and plants, gene therapy, and protein production.

Problems solved by technology

This process is time consuming and tedious.

Method used

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  • Chromosome-based platforms
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Examples

Experimental program
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example 2

[0309] A. Construction of Targeting Vector and Transfection into LMtk-Cells for the Generation of Platform Chromosomes

[0310] A targeting vector derived from the vector pWE15 (GeneBank Accession # X65279) was modified by replacing the SalI (Klenow filled) / SmaI neomycin resistance containing fragment with the PvuII / BamHI (Klenow filled) puromycin resistance containing fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc. Palo Alto, Calif.; SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb NotI fragment from the plasmid pFK161 (SEQ ID NO: 118) containing a portion of the mouse rDNA region was cloned into the NotI site of pWEPuro resulting in plasmid pWEPuro9K (FIG. 2). The vector pWEPuro9K was digested with Spel to linearize and transfected into LMtk− mouse cells. Puromycin resistant colonies were isolated and subsequently tested for artificial chromosome formation via fluorescent in situ hybridization (FISH) (using mouse major and minor DNA repeat sequences...

example 3

Construction of Targeting Vector and Transfection into LMtk− Cells for the Generation of Platform Chromosomes Containing Multiple Site-Specific Recombination Sites

[0325] An example of a selectable marker system for the creation of a chromosome-based platform is shown in FIG. 4. This system includes a vector containing the SV40 early promoter immediately followed by (1) a 282 base pair (bp) sequence containing the bacteriophage lambda attP site and (2) the puromycin resistance marker. Initially a PvuII / StuI fragment containing the SV40 early promoter from plasmid pPUR (Clontech Laboratories, Inc., Palo Alto, Calif.; Seq ID No. 30) was subcloned into the EcoRI / CRI site of pNEB 193 (a PUC 19 derivative obtained from New England Biolabs, Beverly, Mass.; SEQ ID No. 32) generating the plasmid pSV40193. The only differences between pUC19 and pNEB193 are in the polylinker region. A unique AscI site (GGCGCGCC) is located between the BamHI site and the SmaI site, a unique PacI site (TTAATTA...

example 4

Lambda Integrase Mediated Site-Specific Recombination of a RFP Expressing Vector onto Artificial Chromosomes

[0329] In this example, a vector expressing the red fluorescent protein (RFP) was produced and recombined into the attP site residing on an artificial chromosome within LMTK− cells. This recombination is depicted in FIG. 7.

[0330] A. Construction of Expression Vectors Containing Wildtype and Mutant Lambda Integrase

[0331] Mutations at the glutamic acid at position 174 in the lambda integrase protein relaxes the requirement for the accessory protein IHF during recombination and DNA supercoiling in vitro (see, Miller et al. (1980) Cell 20:721-729; Lange-Gustafson et al. (1984) J. Biol. Chem. 259:12724-12732). Mutations at this site promote attP, attB intramolecular recombination in mammalian cells (Lorbach et al. (2000) J. Mol. Biol 296:1175-1181).

[0332] To construct nucleic acid encoding the mutant, lambda integrase was PCR amplified from bacteriophage lambda DNA (cI857 ind ...

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Abstract

Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes provide tractable, efficient and rational engineering of the chromosome for a variety of applications.

Description

RELATED APPLICATIONS [0001] This application is a continuation of and claims priority under 35 U.S.C. § 120 to copending U.S. application Ser. No. 10 / 161,403, filed May 30, 2002, to EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, JOSEPHINE LEUNG, ELENA FLEMING, SANDRA STEWART and JOAN SHELLARD, who are the inventors as originally filed, entitled “CHROMOSOME-BASED PLATFORMS,” which claims benefit of priority under 35 U.S.C. §119(e) to U.S. provisional application Ser. No. 60 / 294,758, filed May 30, 2001, to EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, and JOSEPHINE LEUNG, entitled “CHROMOSOME-BASED PLATFORMS” and benefit of priority to U.S. provisional application Ser. No. 60 / 366,891, filed Mar. 21, 2002, to EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, JOSEPHINE LEUNG, ELENA FLEMING, and SANDRA STEWART entitled, “CHROMOSOME-BASED PLATFORMS.”. [0002] This application is related to U.S. application Ser. No. 11 / 006,076, filed Dec. 6, 2004 by EDWA...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/08C12N15/09A01K67/027A61K31/7088A61K48/00A61P35/00C07K14/005C07K19/00C12N5/10C12N9/22C12N15/63C12N15/85C12N15/90C12Q1/02C12Q1/68G01N33/15G01N33/50G01N33/58G01N33/68
CPCC07K14/005G01N2500/10C12N15/63C12N15/85C12N15/90C12N15/902C12N2795/10322C12N2800/108C12N2800/208C12N2800/30C12N2830/00C12N2830/15C12N2830/40C12N2830/60C12N2830/85C12N2840/20C12N2840/203C12Q1/6897C12N9/22A61P35/00
Inventor PERKINS, EDWARDPEREZ, CARLLINDENBAUM, MICHAELGREENE, AMYLEUNG, JOSEPHINEFLEMING, ELENASTEWART, SANDRASHELLARD, JOAN
Owner GLAXO GRP LTD