Chinese Academy of Agricultural Sciences

Shandong Landsee Genetics Co., Ltd.

Grand Life Science & Technology, Ltd.

Yulian Mu

Kui Li

Zhiguo Liu

Shutang Feng

Shulin Yang

Kui Xu

Zhonglin Tang

Qian Gao

Junhua Fan

Long Wang

Wentao Cui

Qimei Chen

Yang Qiu

Huiming Ju

Xuehui Cai

Lijing Bai

Julang Li

Yinghui Wei

Jianlin Han

Changli Ge

A method for detecting bi-allelic gene edited cells based on CRISPR/Cas12a technology is provided. The method provides crRNA, a nucleotide sequence of which that binds target is SEQ ID NO: 6. The crRNA is transcribed from a transcription template in vitro; the transcription template is a product obtained by annealing a single stranded DNA molecule shown in SEQ ID NO: 10 and a single stranded DNA molecule shown in SEQ ID NO: 13. The method provides crRNA of CD71 gene and constructs a CRISPR/Cas12a system using the same for CRISPR/Cas12a detection, achieving the screening for CRISPR/Cas9 induced CD71 gene bi-allelic gene edited cells. This method is simple, easy, fast, and cost-effective for screening a large number of mutant cells.

Method for detecting bi-allelic gene edited cells based on CRISPR/Cas12a technology

Provided is a double-gene knockout vector system, a method for preparing porcine fibroblasts with both CD163 gene and CD13 gene being knocked-out, prepared porcine fibroblasts, and a method for preparing a gene-edited pig with both CD163 gene and CD13 gene being knocked-out. The vector system of the present disclosure comprises a CD163 gene knockout vector and a CD13 gene knockout vector. The CD163 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, with a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 1-3. The CD13 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 4-6.

Method for preparing porcine fibroblasts with both CD163 gene and CD13 gene being knocked-out

The present invention discloses a method for identifying Wuzhishan miniature pig inbred line by using 145 SNPs. The method for auxiliarily identifying whether a pig to be tested is Wuzhishan miniature pig inbred line, provided by the present invention, comprises the following steps: testing genotypes based on 145 SNP sites of the pig to be tested; if all standards of (1) to (145) are satisfied, the pig to be tested is a candidate for Wuzhishan miniature pig inbred line; if all the standards of the above (1) to (145) are not satisfied, the pig to be tested is a candidate for non-Wuzhishan miniature pig inbred line.

Method for identifying wuzhishan miniature pig inbred line by using 145 SNPs

A method for auxiliary identification of WZSP inbred line and its special primers are disclosed. The method may comprise: testing whether a deoxyribose nucleotide at site 1273 from 5' end of genomic DNA comprising SEQ ID NO: 1 in a test pig is A or G, measuring whether the test pig genotype is GG, GA or AA wherein the test pig with GG genotype is a candidate for the WZSP inbred line and the test pigs with GA genotype and AA genotype are non-WZSP inbred line. The method can be applied to breed WZSP inbred line, which can be used to preliminarily screen all the pigs in test pig group, eliminate non-WZSP inbred line, find out a candidate WZSP inbred line and make a further confirmation in combination with other methods. The method and primers can also be used to judge whether a WZSP on market is counterfeiting or not.

Method for auxiliary identification of inbred line of wuzhishan miniature pig and its special primer

A method for preparing a transgenic animal of simultaneous multiple-gene expression is provided. Additionally, a method for preparing a transgenic embryo, which introduces both phytase gene and human myxovirus resistant gene A into a target embryo, to obtain a transgenic embryo is provided. The transgenic animal of simultaneous multiple-gene expression can be achieved by transplanting the transgenic embryo into the body of a female target animal. A significant advantage of the foregoing methods, among many others, exists in that the simultaneous expression of multiple genes can be achieved in one transgenosis, which provides a convenient method for the preparation of combined-gene transferred animals.

Method for preparing a transgenic animal of simultaneous multiple-gene expression

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Beijing, China

Yulian Mu