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Quick and convenient authentication technology fro transgenic insert locus

An insertion site and transgene technology, applied in the field of transgene insertion site identification, can solve problems such as sequence difficulties, multi-site integration transgene deletion or rearrangement, limb abnormalities, etc.

Inactive Publication Date: 2007-07-11
王铸钢
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the mouse transgenic technology also has huge defects: the transgene is integrated in the genome in a nearly random manner, and the integration of the transgene may lead to three results, one is high expression of the transgene, and the other is that the transgene is not expressed due to the influence of the structure and sequence of the nearby genome or low expression, and the third is that the endogenous gene or other functional elements at the integration site are destroyed, resulting in loss of function (according to Palmiter's statistics on 153 transgenic mouse lines, about 7% of transgenic mouse lines have insertion mutations. Abnormal limbs or lethal homozygous embryos, it is estimated that insertion mutations without appearance phenotypes are more common)
The results show that in most cases the transgene is integrated in multiple copies, sometimes up to hundreds of copies; the transgene is mainly integrated at a single site in the genome in a head-to-tail manner, and only in a few cases Under normal circumstances, multi-site integration, deletion or rearrangement within the transgene may occur, occasionally leading to duplication of genome sequence, deletion and chromosomal translocation, etc.
However, these studies failed to establish an economical and rapid method for the identification of transgene integration sites and to promote its application.
Although a small number of sequences at the transgene / genome integration sites have been obtained by using individual genome library screening methods, plasmid recovery methods, and inverse PCR (inverse PCR), due to time-consuming and labor-intensive, or the characteristics of multi-copy integration of transgenes, intermediate copies It will compete with the copy at the end for PCR, and the impact of the sequence change of the transgene after integration makes it very difficult to obtain the sequence at the transgene / genome integration site
Moreover, the genome project had not yet been implemented at that time, and it was very cumbersome and difficult to determine the location of the transgene and the damaged endogenous gene

Method used

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  • Quick and convenient authentication technology fro transgenic insert locus
  • Quick and convenient authentication technology fro transgenic insert locus
  • Quick and convenient authentication technology fro transgenic insert locus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0189] Example 1: Identification of integration sites in pEGFP-PLAG1 transgenic mice (Strategy 1)

[0190] The pEGFP-PLAG1 transgenic plasmid was constructed by cloning the fragment containing the complete coding frame of PLAG1 and the first 100 bases of the translation initiation codon into the EcoRI site of the pCMV-EGFP (purchased from Clontech) vector, and its structure is shown in Figure 4 Show. After the plasmid was digested with NsiI, a 3.8 kb fragment was recovered for microinjection.

[0191] After analyzing the distribution of restriction sites in the fragment sequence of the above-mentioned transgenic vector, it was determined that Sau3AI was used as the restriction endonuclease E1 to digest the genome of the transgenic mouse, so the following linker DNA was designed and synthesized (Sau3AI linker was formed after the two strands were annealed):

[0192] Linker 1: 5'-GTA ATA CGA CTC ACT ATA GGG CAC GCG TGGTCG ACG GCC CGG GCT GGT-3' (SEQ ID NO: 2)

[0193] Linker 2...

Embodiment 2

[0207] Example 2: Identification of integration sites in pEGFP-PLAG1 transgenic mice (Strategy 2)

[0208] Design and synthesize the following linker DNA (blunt end linker is formed after the two strands are annealed):

[0209] Linker 1: 5'-GTA ATA CGA CTC ACT ATA GGG CAC GCG TGGTCG ACG GCC CGG GCT GGT-3' (SEQ ID NO: 10)

[0210] Linker 2: 5′-PO 4 -ACC AGC CC-N2H-3'(5'-OH with PO 4 Substitution, 3'-OH with N 2 H substitution) (SEQ ID NO: 11)

[0211] Dissolve in TE or water to 50pmol / μl, take 5μl (equal molar number) and mix before use, cool down naturally to room temperature for 10 minutes at 80°C and anneal.

[0212] Analyze the sequence of the transgenic vector shown in Example 1 to find out a series of restriction endonucleases EcoRV, NruI, PmacI, XmnI, PshAI, BstZ17I that do not cut the vector DNA and produce blunt ends.

[0213] Take 5-10 μg of genomic DNA from transgenic mice, digest with EcoRV, NruI, PmacI, and XmnI in sequence (i.e., digest with one enzyme to ina...

Embodiment 3

[0224] Example 3: Establishment of transgenic mouse lines and identification of transgenic mouse integration sites using the universal vector Inte-vector

[0225] The IAP promoter and the gene KIF18A3 (1st to 11th exons) cDNA sequence are cloned between the XhoI and BamHI sites of the multi-cloning site region of the universal vector Inte-vector (shown in sequence SEQ ID NO: 1), and the The transgenic plasmid was constructed, digested with NotI, and the linearized fragment between the two NotI restriction sites was recovered for transgenesis.

[0226] According to the method described in Example 1, the genome of the transgenic mouse was digested with Sau3AI, and after purification, it was ligated with the linker (same as Example 1)

[0227] According to the general vector sequence, PvuII is the restriction endonuclease E2 at the 5′ end of the carrier, and KpnI is the restriction endonuclease E2 at the 3′ end of the carrier. Two enzyme digestion systems are established, and the...

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Abstract

The invention discloses an affirming method of gene-transmitting inserting position, which is characterized by the following: digesting limit internally tangent enzyme; connecting interface; proceeding PCR augumentating; obtaining the genome DNA sequence between carrier and interface; affirming the position of exogenous gene; analyzing causal relationship.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to a method for identifying a transgene insertion site. Background technique [0002] Since the emergence of transgenic technology in the 1980s, it has been widely used in the establishment of animal models of human diseases, in vivo research on gene functions, genetic engineering breeding, animal husbandry production, and biopharmaceuticals. Especially after the completion of the Human Genome Project and many model organism genome sequencing projects, the study of gene function, that is, functional genomics research, has become the biggest opportunity and challenge for medical and life science research in this century and even this century. In vivo system research on gene function, regulation of transcriptional expression, embryonic development, pathogenesis of human diseases and screening of new drugs or treatments is one of the most important technical mean...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63
Inventor 王铸钢
Owner 王铸钢