Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Chromosome landing pads and related uses

A phage and cell technology, applied in the field of sequences, can solve the problems of complex and cumbersome high-efficiency expression

Inactive Publication Date: 2016-05-18
THE SCRIPPS RES INST
View PDF1 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Chromosome landing pads and related uses
  • Chromosome landing pads and related uses
  • Chromosome 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 plasmids were 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...

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides methods for stably integrating and / or expressing one or more recombinant polynucleotides in a host cell. The recombinant polynucleotide is usually integrated into the host genome at some natural chromosomal integration site. The integration can be mediated by homologous recombination or with hybrid recombinases targeted to specific chromosomal locations. Stable integration and strong transcriptional activity of foreign genes are supported by native chromosomal integration sites in the host cell, which are located within or near specific genes in the CHO genome, the 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 the insertion of site-specific recombination sequences (chromosomal landing pads) into these specific chromosomal locations. Further provided are engineered host cells containing chromosomal landing pads or transgenes integrated into natural chromosomal integration sites. Further provided are methods and compositions (eg, kits) for integrating and expressing heterologous polynucleotides in host cells with inserted chromosomal landing pads.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/09
CPCC12N5/0682C12N15/907C12N2510/00C12N2800/107C12N2800/30C12N15/68C12N15/85C12N2320/32
Inventor V·P·莫罗周伟B·坎宁安G·M·伊德尔曼
Owner THE SCRIPPS RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com