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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: 2006-02-02
GLAXO GRP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes artificial chromosomes that can be easily engineered and used in cells. These chromosomes contain specific locations for adding new DNA, and can be introduced into cells using recombinase systems. The artificial chromosomes can also contain other genes and be used to produce therapeutic products in animals and plants. The technical effect of this patent is the creation of a versatile platform for introducing new DNA into cells and creating new animals and plants with specific genetic modifications.

Problems solved by technology

This process is time consuming and tedious.

Method used

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  • Chromosome-based platforms
  • Chromosome-based platforms
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Examples

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example 1

[0298] pFK161

[0299] Cosmid pFK161 (SEQ ID NO: 118) was obtained from Dr. Gyula Hadlaczky and contains a 9 kb NotI insert derived from a murine rDNA repeat (see clone 161 described in PCT Application Publication No. WO97 / 40183 by Hadlaczky et al. for a description of this cosmid). This cosmid, referred to as clone 161 contains sequence corresponding to nucleotides 10,232-15,000 in SEQ ID NO. 26. It was produced by inserting fragments of the megachromosome (see, U.S. Pat. No. 6,077,697 and International PCT application No. WO 97 / 40183). For example, H1D3, which was deposited at the European Collection of Animal Cell Culture (ECACC) under Accession No. 96040929, is a mouse-hamster hybrid cell line carrying this megachromosome into plasmid pWE15 (Stratagene, La Jolla, Calif.; SEQ ID No. 31) as follows. Half of a 100 μl low melting point agarose block (mega-plug) containing isolated SATACs was digested with NotI overnight at 37° C. Plasmid pWE15 was similarly digested with NotI overnigh...

example 2

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 PvuIV / 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 SpeI 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, the...

example 3

Construction of Targeting Vector and Transfection Into LMtk- Cells for the Generation of Platform Chromosomes Containing Multiple Site-specific Recombination Sites

[0324] 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 pNEB193 (a PUC19 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 Smal site, a unique Pacd site (TTAATTAA)...

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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 permit tractable, efficient, 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 EDWAR...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H1/00C12N5/04C12N15/87C12N15/82A61K31/7088A01K67/027A61K48/00A61P35/00C07K14/005C07K19/00C12N5/10C12N9/22C12N15/09C12N15/63C12N15/85C12N15/90C12Q1/02C12Q1/68G01N33/15G01N33/50G01N33/58G01N33/68
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, AMY
Owner GLAXO GRP LTD