Multiple-exchange unmarked site-specific integration transgenic system and application thereof

A transgenic and integrase technology, applied in the field of bioengineering, can solve the problems of long production cycle, high cost, and animal transgenic technology needs to be improved, and achieve the effect of short production cycle

Active Publication Date: 2015-07-15
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, there are still problems of efficiency, safety and stability in the application of animal transgenic technology, especially the transgenic of livestock animals, which has low efficiency, long preparation cycle, genetic instability, long strain breeding cycle and high cost
[0004] Therefore, the current animal transgenic technology still needs to be improved

Method used

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  • Multiple-exchange unmarked site-specific integration transgenic system and application thereof
  • Multiple-exchange unmarked site-specific integration transgenic system and application thereof
  • Multiple-exchange unmarked site-specific integration transgenic system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Construction and activity detection of TALEN vector (first construct) for identifying pR26 site

[0076] The pR26 site sequence was analyzed, and SEQ ID NO: 1 was selected as the TALEN vector recognition splicing sequence. Among them, TALEN1 recognizes TGCCGTAATGCTAATAGC (SEQ ID NO:4), that is, the assembled TALEN1 module sequence is NN-HD-HD-NN-NG-NI-NI-NN-HD-NG-NI-NI-NG-NI-NN-HD .

[0077] TALEN2 recognizes the reverse complementary sequence of CTGTCTCCAGTATCTGA (SEQ ID NO:5), and the sequence of modules for assembling TALEN2 is HD-NI-NN-NI-NG-NI-HD-NG-NN-NN-NI-NN-NI HD-NI -NN.

[0078]The TALEN expression backbone vector of this example was transformed according to the following steps: amplify the CMV-MCS-BGPA sequence on the commercial vector pcDNA3.1(-) and connect it to the T vector to construct CMV-T. Synthesize the TALE-fokI sequence, in which the TALE protein synthesizes 456 nucleotides in the N segment (translation of 152 amino acid residues), 189...

Embodiment 2

[0087] Example 2 pR26 Targeting Vector (Second Construct) Construction

[0088] Design left and right homology arm amplification primers to amplify the homology arms. And the upstream primer of the left homology arm introduces the SalI restriction site, and the downstream primer of the left homology arm introduces the loxp sequence and the NheI and HindIII restriction sites; the upstream primer of the right homology arm introduces the lox2272 sequence and the NotI restriction enzyme site, the downstream primer of the right homology arm introduces the NheI restriction site, and the primer sequence is as follows:

[0089] pR26LA-F: GTC GAC GTCAGTTTGCTCCTTCTCG (SEQ ID NO: 10)

[0090] SalI

[0091] pR26LA-R: GCTAGC AAG AAGCTT ATAACTTCGTATAGCATACATTATACGAAGTTAT

[0092] NheI HindIII loxp

[0093] GACACTAGTTTGGAAGCTA (SEQ ID NO: 11)

[0094] pR26RA-F:

[0095] GCGGCCGC ATAACTTCGTATAAGGTATCCTATACGAAGTTAT TCTGTCTCCAGTATC

[0096] NotI lox227...

Embodiment 3

[0117] Example 3 Transfection Screening and Identification of pR26 Targeted Cells

[0118]Extract endotoxin-free TALENs plasmid and pR26 targeting vector plasmid. The pR26 targeting vector was digested with SalI to linearize it. The TALENs plasmid and the pR26 linearized fragment were co-transfected into Bama pig fetal fibroblasts by nuclear transfer method, and the transfection ratio was 2 μg TALENs: 0.4 μg pR26 targeting vector.

[0119] Divide the transfected cells to 10cm, and use 500μg / ml G418 to screen the cells. After 1 week of screening, positive clones appear, pick the positive clones to a 48-well plate and continue to expand the culture, and change the concentration of 200μg / ml G418 maintain. It should be noted that the lethal concentration of G418 is different for different cell lines, so the lethal concentration of G418 must be measured for the cell lines first. The lethal concentration of G418 for the Bama fibroblast cell line in this example is 500 μg / ml.

[0...

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Abstract

Provided are multiple-exchange unmarked site-specific integration transgenic system and an application thereof, wherein the transgenic system includes: a first construction body which is a TALEN carrier, wherein the TALEN carrier identifies and cuts friendly site sequences; a second construction body including a left homologous arm, a right homologous arm, a selective marker expression cassette, a first integrase recognition sequence loxp and a first integrase recognition sequence lox2272; a third construction body including a targeted exogenous gene, a second integrase recognition sequence loxp' and a second integrase recognition sequence lox2272'; and a fourth construction body including an integrase gene Cre expression cassette. The transgenic system can simply, fast and efficiently achieve site-specific insertion of a targeted exogenous gene in an animal genome, and prepared transgenic animal cells do not contain any resistant marker gene, the preparation cycle is short, preparation is safe and reliable, and the targeted exogenous gene can stably inherited to a next generation.

Description

technical field [0001] The invention relates to the field of bioengineering. Specifically, the present invention relates to a marker-free site-directed integration transgene system capable of multiple exchanges and its application, more specifically, to a transgene system, a transgenic animal cell and a preparation method thereof. Background technique [0002] Non-human transgenic animals can be used as an ideal model for screening and identifying drugs, which can be used to study gene functions, explore the pathogenesis of diseases for humans, seek effective therapeutic approaches, and can also be used for research on the improvement of animal genetic quality. The research on transgenic animals contains huge economic value and has very broad application prospects. At present, it has become one of the investment hotspots of bioengineering in the world, and has also promoted the development of transgenic technology research. [0003] At present, animal transgenic technology ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 张欢欢李勇刘楚新刘欢张兴举李飞达杜玉涛
Owner 深圳华大基因农业控股有限公司
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