Site-specific integration method of exogenous genes

An exogenous gene, site-specific integration technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, enzymes, etc., can solve the problems of low production efficiency of transgenic animals, and achieve the elimination of safety concerns, cost savings, and avoidance of express the effect of silence

Active Publication Date: 2014-07-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The efficiency of transgenic animal production using conventional gene targeting is very low, only 10 -6 -10 -7 The efficiency (Hanson K D et al.1995), and the final c

Method used

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  • Site-specific integration method of exogenous genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1, the construction of TALENs expression vector

[0084] 1. Screen the target area

[0085] Based on a large number of analysis, screening and verification, it was confirmed that a sequence on the second exon of the bovine β-casein gene (AC_000163) was used as the target. The target sequence is as follows:

[0086] 5'- TGAGAGCCATGAAGGT CCTCATCCTTGCCTGCCTG GTGGCTCTGGCCCTTGC -3' (SEQ ID No. 1)

[0087] The underlined part in SEQ ID No.1 is the part that can be specifically bound by the binding domain in the TALENs protein, and the middle part of the binding site is the FokI endonuclease cleavage recognition site.

[0088] 2. Synthesis of TALEs protein

[0089] Based on the target sequence shown in SEQ ID No.1, a pair of TALE proteins (TALE protein-I and TALE protein-II) were designed.

[0090] (1) The coding genes for synthesizing TALE protein-I and TALE protein-II are respectively shown in SEQ ID No.2 (positions 4 to 1533 from the 5' end of SEQ ID No.2 ...

Embodiment 2

[0104] Embodiment 2, the construction of gene targeting vector

[0105] 1. Amplification and sequencing of homology arms of β-casein gene sequence

[0106] (1) According to the bovine β-casein sequence published by GeneBank (Genebank number: NW_003103978.1), the upstream primer 1 and downstream primer 1 were designed to amplify part of the homologous left arm of the β-casein gene sequence.

[0107] Upstream primer 1: 5′-CGG GGTACC AGAGATGTTGATGGCAAGAAC-3'; (SEQ ID No. 6)

[0108] (The underlined sequence is the KpnI restriction recognition site)

[0109] Downstream Primer 1: 5′-GC GTC GAC CTCTCAATTCCTGGGAATGGG-3'. (SEQ ID No. 7)

[0110] (The underlined sequence is the recognition site for SalI digestion)

[0111] (2) Design upstream primer 2 and downstream primer 2 to amplify the homologous right arm of part of the β-casein gene sequence.

[0112] Upstream primer 2: 5′-ATTT GCGGCCGC TTGCAAGAGAGGTAAATACAG-3'; (SEQ ID No. 8)

[0113] (The underlined sequence is the...

Embodiment 3

[0135] Example 3. Site-directed integration of human lysozyme gene at the β-casein locus in bovine fetal fibroblasts

[0136] Fixed-point integration strategies such as Figure 7 shown.

[0137] 1. Cell co-transfection of targeting vector pL452-LYZ and TALENs plasmid

[0138] Cell transfection was performed using amaxa nucleofector. The specific process is as follows:

[0139] (1) Digest and collect 094 bovine fetal fibroblasts from two bottles of T25 cell bottles (the number of cells in each bottle is about 8×10 6 ) and 3ug SacⅡ-digested linearized targeting vector pL452-LYZ and 6ug TALENs plasmids (3ug each of TALENs-LF6 and TALENs-RF2) and 400μl Nucleofector reagent were mixed to obtain a mixture, which was divided into 4 electric shock cups for Electric shock, and then respectively culture the mixture after electric shock in four T-25 petri dishes.

[0140] (2) After 48 hours, the cultured cells in each culture dish were digested and spread into 15 10cm dishes respect...

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Abstract

The invention discloses a site-specific integration method of exogenous genes, and particularly discloses a method for integrating the exogenous genes into second exons of bovine beta-casein genes in a site-specific manner. The method comprises the following steps: expressing TALE (Transcription Activator Like Effector) protein-I and TALE protein-II in isolated bovine cells so as to obtain cells, the target sequences of second exons of which change suddenly; meanwhile, recombining the exogenous genes into the target sequences in a homologous manner so as to realize the site-specific integration of the exogenous genes on the second exons of the bovine beta-casein genes. The method has the advantages that human lysozyme genes or other target genes are capable of transcribing the site-specific integration of very active beta-casein gene loca in mammary gland, the location effect is overcome, the target gene expression silencing is avoided, the relatively high expression level of the human lysozyme or other recombinant protein in mammary gland can be realized, the success rate of transgenic breeding is increased, and meanwhile, the cost of producing and purifying the recombinant protein in a later period can be saved.

Description

technical field [0001] The invention relates to a method for site-specific integration of exogenous genes. Background technique [0002] Lysozyme is an important antibacterial factor in human milk. It has a killing effect on most Gram-positive bacteria and some Gram-negative bacteria. It plays an important role in enhancing the immunity of infants and young children. It is also widely used in light industry and food industry. In 2009, the U.S. FDA approved the world's first anti-thrombin transgenic drug produced by mammary gland bioreactor, which marked that the use of transgenic animals to produce recombinant proteins has truly entered the stage of industrialization. [0003] The use of mammary gland bioreactors to produce recombinant proteins has broad market application prospects, but the traditional transgenic technology still has some shortcomings in the research of mammary gland bioreactors. In traditional transgenic research, due to the random insertion of exogenous...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/11C12N9/36
Inventor 戴蕴平王本利丁方荣李松王海萍郑敏李京李玲
Owner CHINA AGRI UNIV
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