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Pig ROSA26 specific integration site and application thereof

An integration site and specific technology, applied in the field of genetic engineering, can solve the problems of rare transgenic large animals, lack of foreign gene integration sites, etc., and achieve the effect of efficient and specific targeting

Inactive Publication Date: 2013-01-02
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, except for mice, there are few transgenic large animals obtained by gene targeting, and there are no related reports in transgenic pigs, mainly due to the lack of ideal exogenous gene integration sites

Method used

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  • Pig ROSA26 specific integration site and application thereof
  • Pig ROSA26 specific integration site and application thereof
  • Pig ROSA26 specific integration site and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Pig ROSA26 specific integration site

[0026] The pig ROSA26-specific integration site sequence is shown in SEQ ID NO.1, or under stringent conditions (Tm-10~15°C), it can hybridize with the DNA sequence defined in SEQ ID NO.1 and can promote the transcription and translation of the target gene DNA sequence, or a DNA sequence that is more than 90% homologous to the DNA sequence defined by SEQ ID NO.1 and that can promote the transcription and translation of the target gene.

Embodiment 2

[0027] Example 2 Obtaining of porcine ROSA26-specific integration sites

[0028] Design PCR primers based on the existing sequences on both sides of the gap, the sequence is: 5'-GGATCTAATTGGAGCTATAACTGCCAGC-3'(forward)5'-GCTGAGGGTCCCAAATGCTTTG-3'(reverse);

[0029] The PCR conditions are: 94°C for 3min; 94°C for 30sec, 60°C for 30sec, 72°C for 1min (30cycles); 72°C for 10min; 4°C for 1h.

[0030] The Rosa26 locus is located on the pig chromosome 13. Most of the sequence information has been given by the NCBI Sscrofa10.2 database, but 108 bp is missing at the 3' end of the first intron. Design primers based on the sequence information: obtain the missing sequence information by PCR, obtain the entire sequence of the promoter, first exon, first intron and second exon of the porcine Rosa26 gene, and apply for patents for these sequences , protected from mediating the expression of exogenous or endogenous genes, characterized by their DNA sequence. as follows:

Embodiment 3

[0031] Example 3 Targeting vector based on porcine ROSA26-specific integration site

[0032] Targeting vector based on porcine ROSA26-specific integration site ( image 3 ), its nucleotide sequence is shown in SEQ ID NO.2.

[0033] The construction method is as follows: use PPNT6 (company addgene, product number 11072) as the backbone to construct the porcine ROSA26 targeting vector.

[0034] PCR clone ROSA265' and 3' homology arms, the size is 1.4kb and 3.6kb respectively, the amplification primers are:

[0035] 5' homology arm, 5'-CGGGAGTGCGGCCCGCCCTGCGGC-3' (forward)

[0036] 5'-AGTAGATCCGTGCTTTTTAACCTATC-3'(reverse);

[0037] 3' homology arm, 5'-GAGTTTTACAGTCATCCCATTTGTAGACTTTTGC-3'(forward)

[0038] 5'-AGCTCTGATCCCGTGT TGTTGTGGCATAG-3' (reverse).

[0039] The 5' homology arm was connected at the NotI and KpnI restriction site, and the 3' homology arm was connected at the XhoI and NheI restriction site. Then, the pR26, CMV and EF1a promoters are connected into the Cla...

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Abstract

The invention discloses a pig ROSA26 specific integration site and an application thereof. The nucleotide sequence of the site is represented by SEQ ID NO.2. A targeting vector derived from the site is represented by SEQ ID NO.1. The ROSA26 specific site adopted by the invention is not subjected to immune-mediated exclusion and epigenetic modification in genomes of pigs or higher mammals. The site can mediate stable and efficient expression of exogenous genes. With the ROSA26 specific site targeting vector, targeting can be realized with high efficiency.

Description

technical field [0001] The invention relates to a porcine ROSA26 specific integration site, which belongs to the field of genetic engineering. Background technique [0002] In the past ten years, transgenic technology has been rapidly developed and widely used. Its combination with cloning technology has made it possible for the production of transgenic animals to move from basic research to industrialization. Stable, efficient and extensive expression in transgenic animals. Current research shows that this is mainly related to the integration site of the foreign gene in the host genome. Therefore, an integration site that enables efficient integration and stable expression of the foreign gene is obtained is very important. [0003] In mice, it has been proved that the ROSA26 site can mediate the expression of exogenous genes extensively and efficiently in almost all cells, whether in various embryonic developmental stages or in various adult tissues and organs, and has bee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63A01K67/027
CPCA01K67/0275C12N15/8509A01K2217/07A01K2227/108C12N15/907C12N15/79C07H21/04C12N15/85
Inventor 孔庆然武美玲刘忠华
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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