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Methods for engineering host cell genomes and uses thereof

A host cell and gene technology, applied in the fields of cell biology, bioengineering, and molecular biology, can solve the problems of difficult screening, increased uncontrollable uncertainty of cell lines, and heterogeneity of cell lines

Active Publication Date: 2022-07-05
ANTAGEN BEIJING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After several more genes are transferred, the heterogeneity of the cell line will be caused, and the screening is difficult
Moreover, due to the gene silencing effect (Gene Silencing), genes inserted in different chromosomal sites will slowly turn off expression at different speeds, which increases the uncontrollability of cell lines and the uncertainty of experiments
[0012] 2) If each gene uses a carrier with a specific drug resistance gene, then after 3 to 4 genes are transferred, the existing drug resistance gene will be almost used up

Method used

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  • Methods for engineering host cell genomes and uses thereof
  • Methods for engineering host cell genomes and uses thereof
  • Methods for engineering host cell genomes and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] Example 1: Construction of CHO cell targeting vector and establishment of anchor cell line

[0209] In order to achieve continuous, cyclical, and unlimited site-directed integration in the CHO cell genome, it is first necessary to find a high-expression hot spot (Hot-Spot) in the CHO cell genome. After in-depth research, the inventor found that the promoter (Promoter) of the highly constitutively expressed housekeeping gene GAPDH meets the requirements of open chromosome structure and constant high expression. For example, Bahr S. ​​et al. identified a site similar to mouse ROSA26 in CHO cells. ROSA26 is a known structurally open and constant high expression site in the mouse genome, and is often used to implant foreign genes to prepare transgenic mice. Although the ROSA26 gene also exists in CHO cells, the transcriptional intensity of its mRNA level is lower than that of several other invariant housekeeping genes, especially only 1 / 300 of the transcriptional intensi...

Embodiment 2

[0215] Example 2: Construction of "Toggle-In" vector and method for stable co-expression of multiple genes

[0216] In order to solve the problem of stable co-expression of multiple genes in the transformation of CHO cell lines, the inventors developed a set of "Toggle-In" platform for Cre-LoxP-mediated homologous recombination based on the aforementioned anchored CHO cell lines. system. The feature of this system is that it can achieve continuous, cyclical, and unlimited genome site-directed integration with two vectors (pTOG3 and pTOG4) in sequence and only two drug resistance genes (hygromycin B and puromycin). , these CHO cell clones, each of which is isogeneic, can achieve 1:1 expression of the insert.

[0217] pTOG3 (its nucleotide sequence is shown in SEQ ID NO: 11, which carries the DsRed-E2 tracer protein, and its nucleotide sequence is shown in SEQ ID NO: 36) and pTOG4 (its nucleotide sequence is shown in SEQ ID NO: 12, which carries the d1EGFP tracer protein, an...

Embodiment 3

[0229] Example 3: Knockout of Fut8 gene in CHO cells using TALEN technology

[0230] In order to conveniently and quickly knock out the Fut8 gene in CHO cells, the inventors constructed a pair of TALEN vectors targeting the 10th exon of the CHO cell Fut8 gene (the DNA sequence encoding the left arm of the TALEN protein for knocking out the Fut8 gene in CHO cells is shown in SEQ ID NO. : 18, the DNA sequences encoding the right arm of the TALEN protein that knocked out the Fut8 gene in CHO cells are shown in SEQ ID NO: 19), named FL#2 and FR#12, respectively. 5 μg of the plasmids prepared by the two miniPrep were taken and introduced into 1 million CHO-K1 cells (ATCC, CCL-61) by electroporation. The electroporator is the Neon Electroporation system from LifeTech. Electric transfer conditions are 1620v, 10ms, 3 pulses. After electroporation, CHO cells were cultured in DMEM medium containing 5% FBS for 8 days without any drug selection. This growth stage allows those clones ...

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Abstract

The present invention provides a method for integrating multiple exogenous genes at specified sites in the host cell genome. The present invention also provides a method for modifying the host cell genome, the method comprising: a. integrating an exogenous gene at a designated site in the host cell genome; and / or b. knocking out fucose transfer in the host cell genome Enzyme 8 gene and / or glutamine synthase gene and / or α2,3-sialyltransferase gene 4 and / or α2,3-sialyltransferase gene 6. The present invention also provides host cells prepared according to the methods of the present invention. The present invention also provides the use of the host cell for producing the target protein. The cell genome of the CHO strain provided by the present invention integrates genes capable of improving protein expression at a specific point, which is of great significance for the development of novel antibodies and high-end imitation antibodies.

Description

technical field [0001] The present invention belongs to the fields of molecular biology, cell biology and bioengineering. In particular, the present invention relates to a method for modifying the genome of a host cell, the present invention also relates to the use of a host cell modified by the method, and the use of said modified host cell. Background technique [0002] Since the US FDA approved Genetech's recombinant insulin in 1982, the recombinant protein has been successfully used in scientific research, diagnosis, treatment and prevention of various diseases. Currently, recombinant proteins are mainly produced and expressed using exogenous protein expression systems. The production of recombinant proteins from mammalian cells has become the mainstream technology for biotech drugs. Most of the protein drugs currently on the market, undergoing clinical trials and preclinical research are expressed by mammalian cells, and among them, Chinese Hamster Ovary (CHO) cells a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/85C12N5/10
CPCC12N15/907C12N15/85C12N5/0682C12N2800/107C12N2800/30C12N2510/02
Inventor 高闻达毛昌群邵静岳国华
Owner ANTAGEN BEIJING BIOTECH CO LTD
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