Pig specific friendly site Pifs501 and application thereof

Abstract

The invention relates to the field of genetic engineering and particularly discloses a pig specific friendly site Pifs501. The position of the friendly site Pifs 501 in a genome is 44288819bp on the chromosome 16 (NC_010458.3, based on the pig genome sequence information version number Sscrofa10.2, September, 2011). The pig specific friendly site Pifs501 can be used for stable expression of exogenous genes, and the problems of instable expression of the exogenous genes and unpredictability of the position effect during transgenosis are solved. By means of the pig specific friendly site Pifs501, fixed-site exogenous gene integration to livestock becomes reliable, and the foundation for fixed-site integration of the livestock is laid. A targeting vector provided on account of the pig specific friendly site Pifs501 can achieve targeting efficiently and specifically.

Description

technical field

[0001] The invention relates to the field of genetic engineering, in particular to the pig-specific friendly site Pifs501. Background technique

[0002] In the production process of transgenic animals, whether foreign genes can be expressed efficiently and stably in transgenic animals is the key to transgenic technology. The exogenous gene is randomly integrated in the genome of the recipient animal, and the expression level will be unstable due to the position effect, and the transgene will be silenced by the epigenetic modification. In order to achieve sustained and stable expression of exogenous genes, site-specific transgenesis can solve various problems caused by position effects. In recent years, ZFN and TALEN technologies, as well as the emerging CRISPR / Cas9 technology, have provided new tools for site-specific integration and gene knockout of genes, improved the efficiency of transgenics, and have been successful in many species. However, one of the...

Examples

Embodiment 1

[0036] Example 1 is used to illustrate the pig-specific friendly site of the present invention.

[0037] The position of the friendly site Pifs501 on the genome is: bp 44288819 on chromosome 16 (NC_010458.3, based on the pig genome sequence information version number Sscrofa10.2, September 2011).

Embodiment 2

[0039] Example 2 is used to illustrate the sgRNA specifically targeting the friendly site Pifs501 of the present invention, the sgRNA is transcribed from the DNA sequence shown in SEQ ID No.1.

Embodiment 3

[0041] Example 3 is used to illustrate the CRISPR-Cas9 targeting vector for the friendly site Pifs501 of the present invention.

[0042] The construction method of CRISPR-Cas9 targeting vector is as follows:

[0043] 1. Construction of targeting vector

[0044]Complementary pairing of the sequence shown in SEQIDNo.1 and the sequence shown in SEQIDNo.2 to construct double-stranded DNA; the construction process is as follows: 94°C, 5min, then 35°C, 10min, and then put it on ice immediately, for oligonucleotide to save.

[0045] The px330 backbone vector was digested with the restriction endonuclease BbsI overnight, and after recovery, it was ligated with the annealed oligonucleotide at 16°C for 3 hours. Transformation and coating are carried out by conventional transformation methods. After a single colony grows, several colonies are picked for expansion and sequenced. The sequencing verification was correct, and the successfully constructed CRISPR-Cas9 targeting vector was ...

technical field

[0001] The invention relates to the field of genetic engineering, in particular to the pig-specific friendly site Pifs501. Background technique

[0002] In the production process of transgenic animals, whether foreign genes can be expressed efficiently and stably in transgenic animals is the key to transgenic technology. The exogenous gene is randomly integrated in the genome of the recipient animal, and the expression level will be unstable due to the position effect, and the transgene will be silenced by the epigenetic modification. In order to achieve sustained and stable expression of exogenous genes, site-specific transgenesis can solve various problems caused by position effects. In recent years, ZFN and TALEN technologies, as well as the emerging CRISPR / Cas9 technology, have provided new tools for site-specific integration and gene knockout of genes, improved the efficiency of transgenics, and have been successful in many species. However, one of the...

Examples

Embodiment 1

[0036] Example 1 is used to illustrate the pig-specific friendly site of the present invention.

[0037] The position of the friendly site Pifs501 on the genome is: bp 44288819 on chromosome 16 (NC_010458.3, based on the pig genome sequence information version number Sscrofa10.2, September 2011).

Embodiment 2

[0039] Example 2 is used to illustrate the sgRNA specifically targeting the friendly site Pifs501 of the present invention, the sgRNA is transcribed from the DNA sequence shown in SEQ ID No.1.

Embodiment 3

[0041] Example 3 is used to illustrate the CRISPR-Cas9 targeting vector for the friendly site Pifs501 of the present invention.

[0042] The construction method of CRISPR-Cas9 targeting vector is as follows:

[0043] 1. Construction of targeting vector

[0044]Complementary pairing of the sequence shown in SEQIDNo.1 and the sequence shown in SEQIDNo.2 to construct double-stranded DNA; the construction process is as follows: 94°C, 5min, then 35°C, 10min, and then put it on ice immediately, for oligonucleotide to save.

[0045] The px330 backbone vector was digested with the restriction endonuclease BbsI overnight, and after recovery, it was ligated with the annealed oligonucleotide at 16°C for 3 hours. Transformation and coating are carried out by conventional transformation methods. After a single colony grows, several colonies are picked for expansion and sequenced. The sequencing verification was correct, and the successfully constructed CRISPR-Cas9 targeting vector was ...

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