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Method for positioning and integrating transgenosis and application thereof

A gene and exogenous gene technology, applied in the field of transgene positioning and integration, can solve problems such as breeding difficulties, excessively high integration copy number, and changes in transgene expression traits.

Active Publication Date: 2013-10-23
SHANGHAI TRANSGENIC RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The random integration of exogenous genes may lead to the following problems: 1. The probability of transgene integration into inactive chromosomal sequences is higher than that of active chromosomal regions. Due to the influence of integration sites, the expression of most transgenes is low or not expressed; 2. Transgene integration When high-frequency recombination sites are inserted, genetic instability will occur; 3. Random exogenous gene integration often causes endogenous gene mutation or expression changes, affecting the normal development and health of transgenic animals; 4. Multi-site integrated transgenic animals, Separation of integration sites in offspring leads to changes in transgene expression traits and breeding difficulties; 5. Too high or too low integrated copy number can easily lead to low or no expression of transgenes
However, there is currently no method for controlling the efficient integration of the target gene at a specific location in the genome, so there is an urgent need in this field to develop an efficient method for the positioning and integration of transgenes

Method used

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  • Method for positioning and integrating transgenosis and application thereof
  • Method for positioning and integrating transgenosis and application thereof
  • Method for positioning and integrating transgenosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Obtaining loxp localization integration cell line

[0099] 1. Isolation of Human Lysozyme Transgenic Goat Fetal Fibroblasts

[0100] On the 32nd to 40th day after mating between a normal commercially available female goat and a ram containing the wild-type Loxp element (the ram is disclosed in patent ZL200510110772.1), the fetus is removed by surgical operation and transferred to a super-clean workbench, and the obtained Wash the fetus twice with PBS buffer containing 1% double antibody (penicillin 100U / ml, streptomycin 0.1mg / ml), remove the head, limbs and viscera, and soak the remaining tissues in 75% ethanol for 30s; Wash fetal tissue 3-5 times with PBS without double antibody, then cut into 1mm 3 Tissue pieces of different sizes were laid flat on a 100mm cell culture dish, and the tissue pieces were evenly spread out, and the GMEM culture medium (purchased from Invitrogen Company of the United States) added with 10% FBS, penicillin and non-essential amino...

Embodiment 2

[0113] Example 2 Expression and purification of TAT-Cre enzyme

[0114] Pick the expression plasmid pET 28.2TAT-Cre containing TAT-Cre recombinase (this vector can be freely obtained from Howard Hughes Medical Institute, and the vector structure, multiple cloning sites, complete nucleic acid sequence and other information are from http: / / cmm.ucsd.edu / Lab_Pages / dowdy / Vectors / vector_seq / pET28.2bTATCRE.txt ). The colony containing the TAT-Cre recombinase expression plasmid was inoculated in 20ml of LB medium containing kanamycin, and the bacteria were shaken overnight at 37°C. Inoculate the overnight bacterial solution containing the target plasmid in 2L kana LB medium at a ratio of 1:100, shake the bacteria for 3 hours at 37°C. Add IPTG to induce expression, make the final concentration 1mmol / L, shake the bacteria for 3h at 37°C. Centrifuge at 3000×g for 10 minutes to harvest the cells. Resuspend the cells in 100ml pre-cooled PBS, centrifuge at 5000×g for 10 min, and harv...

Embodiment 3

[0119] Example 3 Introduction of Loxp mutation sites and identification

[0120] One side of the full artificial synthesis is a mutant Loxp site, the other side is a wild-type homo-directional Loxp site, and the middle is the full sequence of the expression framework of the screening marker genes neo and TK (sequence shown in SEQ ID NO.: 31) . It is cloned into the SalI site of the commercially available pGEM-7Z vector, and the recombinant plasmid is named pGEM-2loxp'. Using the TAT-Cre-mediated loxp recombination method obtained in Example 2, this element was introduced into human lysozyme transgenic goat fetal fibroblasts, and the selection marker across the wild-type homoloxp site in the cells was replaced. The studies on the positioning and integration of the transgenic components carried out below in this paper all use the human lysozyme transgenic goat fetal fibroblasts with the mutated loxp site.

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Abstract

The invention relates to a method for positioning and integrating transgenosis and an application of the method, and particularly provides a variant loxP element. The method is characterized in that a sequence 'ATAAC' of an inverted repeat sequence in a wild type loxP locus mutates into 'CACCT', and thus the variant loxP element can be obtained. The variant loxP element can greatly improve the integration specificity and integration efficiency of a cre-loxP system, and therefore, efficient local integration of genes can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for positioning and integrating a transgene and its application. Background technique [0002] The method of preparing transgenic animals plays a very important role in basic research and applied research. At present, scientists have carried out a variety of transgenic technologies to improve the targeting and integration efficiency of transgenic animals. However, in large animals, the integration efficiency and expression rate of transgenes are not ideal. [0003] The current international general transgenic method is microinjection, which has high cost and low success rate. As far as transgenic animals—mammary gland bioreactors are concerned, the success rate is only about 3%. Microinjection leads to random integration of foreign genes on chromosomes, and the expression of transgenes is greatly influenced by the surrounding host genome. The random...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N5/10A01K67/00
Inventor 成国祥刘思国陈建泉徐旭俊刘国辉
Owner SHANGHAI TRANSGENIC RES CENT
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