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Chromosomal landing pads and related uses

A chromosome and bacteriophage technology, applied in the direction of stably introducing foreign DNA into chromosomes, vectors, nucleic acid vectors, etc., can solve the problems of cumbersome, high-efficiency expression, etc.

Inactive Publication Date: 2014-02-19
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, obtaining stable, high-efficiency expression of integrated transgenes remains complex and cumbersome, requiring a large number of clones to be screened to select the desired integrating cells

Method used

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  • Chromosomal landing pads and related uses
  • Chromosomal landing pads and related uses
  • Chromosomal landing pads and related uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Identification of native chromosomal loci with strong transcriptional activity

[0096] Plasmid for random integration into the CHO genome: modified based on the original attP-containing plasmid described by Thyagarajan et al. (2001, Mol Cell Biol. 21(12):3926-34). Specifically, the original plasmid was modified to replace the Zeocin marker with a neomycin marker. In addition, the firefly luciferase gene was replaced with the EGFP gene controlled by the SV40 promoter ( figure 1 ).

[0097] Stable transfection: The modified plasmid was purified using Qiagen Midiprep columns. 25 µg of DNA was digested overnight with the restriction enzyme BamHI to linearize the plasmid. The resulting linear DNA was then transfected into CHO cells to generate stable integration. Two days after transfection, the cells were split into new culture plates and hygromycin was added to the medium to select for stable integration events. Cell cultures were grown for 3 weeks and t...

Embodiment 2

[0105] Example 2 Insertion of landing pads at identified natural chromosomal integration sites

[0106] Homologous recombination: Genomic DNA flanking the integration site is identified and cloned into a plasmid containing positive and negative selection markers ( figure 2 ). For each site, a longer homology arm (3-4 kb in length) was cloned 5' of the neomycin resistance gene. A short homology arm (1.5-2 kb in length) was cloned 3' of the neomycin resistance gene. A single phage-binding site, attP, is located at the end of the long homology arm.

[0107] The homologous recombination plasmid was digested with NotI enzyme to linearize the plasmid; the long homology arm was at one end of the linear DNA. After transfection into CHO cells, neomycin was added to the medium to select for cells that integrated the linear DNA in the cell chromosome. A pool of resistant clones was obtained after 4-6 weeks. The cells are then negatively selected by adding ganciclovir to the mediu...

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Abstract

Provided herein are methods for stable integration and / or expression of one or more recombinant polynucleotides in a host cell. The recombinant polynucleotides are typically integrated into the host genome at some native chromosomal integration sites. The integration can be mediated by homologous recombination or by using a hybrid recombinase targeting the specific chromosomal locations. The native chromosomal integration sites in the host cells, which support stable integration and strong transcription activities of foreign genes, are present within or adjacent to specific genes in the CHO genome, ankyrin 2 gene (Ank2), cleavage and polyadenylation specific factor 4 gene (Cpsf4), C-Mos gene, and Nephrocystin-1 / Mal gene. Also provided are methods and nucleic acid molecules for inserting site-specific recombination sequences (chromosomal landing pads) into these specific chromosomal locations, engineered host cells containing chromosomal landing pads, methods and compositions (e.g., kits) therefore.

Description

[0001] References to Priority Documents [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 61 / 516,612, filed April 5, 2011. This application claims priority on the above filing date, and the disclosure of the above provisional patent application is hereby incorporated by reference in its entirety. [0003] Inclusion of sequence listing [0004] This application includes an electronic sequence listing equivalent to a paper sequence listing, electronically submitted through the EFS website, and a computer-readable sequence listing electronically submitted through the EFS website, including a file named "37651505001WOSEQUENCELISTING.txt", This file is 213476 bytes in size and was created on April 5, 2012, and these sequence listings are incorporated herein by reference in their entirety. Background technique [0005] Heterologous polynucleotides are routinely integrated into the genome of mammalian cells for th...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/09
CPCC12N15/907C12N2800/30C12N2800/107C12N5/0682C12N2510/00C12N15/68C12N15/85C12N2320/32
Inventor V·P·莫罗周伟B·坎宁安G·M·伊德尔曼
Owner THE SCRIPPS RES INST
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