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Method of recombination and agents useful for same

A homologous recombination and inhibitor technology, applied in the field of recombining circular nucleic acid sequences and linear nucleic acid sequences and modifying bacterial artificial chromosomes, can solve problems such as the degree of homologous recombination that cannot be well controlled

Inactive Publication Date: 2002-01-16
THE MURDOCH INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In most of these methods, the degree of homologous recombination is not well controlled, so there is a risk of unwanted rearrangements

Method used

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  • Method of recombination and agents useful for same
  • Method of recombination and agents useful for same
  • Method of recombination and agents useful for same

Examples

Experimental program
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Effect test

Embodiment 1

[0117] Example 1 Inducible recE pathway for homologous recombination of PCR fragments to circular substrates in E. coli DH10B cells

[0118] The recE pathway for homologous recombination between linear and circular substrates is active in sbcA recBC strains JC8679 and JC9604 42-45 . The pathway is independent of recA 46 , because the recT gene seems to fulfill the role of recA in pairing with homologous sequences 47 . In these cells, kanamycin resistance (Km r ) PCR fragment of gene (PCRkan50) was precisely homologously recombined into chloramphenicol resistance (Cm r ) in the backbone of the pEBAC140 vector, resulting in pEBAC140::kan50. However, it was not possible to transfer the 200 kbp EBAC / 148-globin clone in DH10B cells to JC9604 electrocompetent cells for subsequent modification, presumably due to host-restricted differences between the two strains. Since BAC / PAC are normally maintained in DH10B cells, any modification can be performed directly in DH10B cells har...

Embodiment 2

[0121] Example 2 Homologous recombination of PCRkan50 and PCRkan140 into the vector backbone of the 200 kb β-globin clone pEBAC / 148 in DH10B cells

[0122] We attempted to modify the 200 kb β-globin clone pEBAC / 148 using PCRkan140 to see if the same method could be used to modify a large, single-copy BAC clone. pGETrec was electroporated into DH10B (pEBAC / 148) cells. Electrocompetent cells were prepared using clone DH10B (pGETrec, pEBAC / 148) carrying both plasmids. Cells were induced with L-arabinose for 40 minutes, and then collected to prepare electrocompetent cells. Electroporation of PCRkan140 into these cells yielded over 1000 Cm per electroporation r Km rRecombinants (Table 1, Experiments 3, 4). When the cells were induced with L-arabinose at the beginning of the culture before the preparation of electrocompetent cells, no Cm r Km r Recombinant. Under L-arabinose induction, prolonged overexpression of recE, recT and gam genes was toxic to DH10B cells. Cells harbo...

Embodiment 3

[0129] Example 3 Construction of pEBAC / 148

[0130] The second generation pEBAC140 BAC / PAC cloning vector (Figure 1) pools the first generation PACs 24 and BAC 23 Multiple features of cloning systems, and HAEC systems 48 OriP and EBNA-1 from Epstein-Barr virus. It also contains several other features, including polyclonal regions of rare cuts. pEBAC / 148 (Fig. 2) was made from pEBAC140 by reverse ligation of a 185 kb genomic fragment carrying the entire β-globin locus using a method based on the PAC cloning protocol. Briefly, the RPCI 1 human total genome PAC library was screened by PCR primers derived from the 5'- and 3'-ends of the β-globin locus 49 . A PAC clone (PAC148 / β-globin) carrying a 185 kb genomic insert was identified and confirmed to contain the complete β-globin locus (about 73 kb), as well as additional sequences at the 5' and 3' ends. The 185 kb genomic insert was separated as a single fragment by NotI digestion, pulsed field gel electrophoresis ...

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Abstract

The present invention relates generally to a method for the recombination of at least two nucleic acid sequences and agents useful for same. More particularly, the present invention contemplates a method of recombining, in a host cell which expresses the recBCD nuclease or a functional derivative thereof, a circular nucleic acid sequence with a linear nucleic acid sequence. The method of the present invention is useful, inter alia, for the modification of bacterial artificial chromosomes by homologous recombination with a linear DNA sequence.

Description

field of invention [0001] The present invention generally relates to methods of recombining at least two nucleic acid sequences and reagents therefor. More specifically, the present invention relates to methods for recombining circular and linear nucleic acid sequences in a host cell expressing a recBCD ribozyme or a functional derivative thereof. The method of the invention can be used in particular for the modification of bacterial artificial chromosomes by homologous recombination with linear DNA sequences. Background of the invention [0002] A detailed bibliography of the numerical references in this specification is centrally listed at the end of the specification. [0003] It is necessary to further understand the structure and composition of higher eukaryotic genomes. Genomic sequences are often much longer than coding sequences. Although the functions of most non-coding sequences are still unknown, they clearly play important roles in regulating gene expression, ...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K48/00G01N33/50C07H21/04C12N1/21C12N9/22C12N15/09C12N15/10C12N15/90
CPCC12N15/902C12N9/22C12N15/10
Inventor P·艾欧安诺K·纳拉亚南
Owner THE MURDOCH INST
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