Title:
METHOD FOR BUILDING SEVERE IMMUNODEFICIENCY AND LIVER DAMAGE DUAL PIG MODEL AND APPLICATION
Document Type and Number:
WIPO Patent Application WO/2023/077360
Kind Code:
A1
Abstract:
A method for building a severe immunodeficiency and liver damage dual pig model and an application, relating to the technical field of animal biology. In the method, a CRISPR/Cas9 technique is used to knockout RAG2, IL2RG and FAH genes in porcine fetal fibroblasts, a somatic cell nuclear transfer technique is used to construct a RAG2-/-/IL2RG-/Y/FAH-/-three gene-edited cloned pig, and a severe immunodeficiency and liver damage dual pig model is obtained by means of phenotypic analysis and identification. By means of the method, the problems in existing model building technology such as a long production cycle, low efficiency, irreversible damage, lack of immunodeficiency and liver damage, and a nonideal humanization degree in application are overcame, and severe immunodeficiency and liver damage dual pig models can be built in batches by means of a continuous cloning technique, such that the present invention has great advantages and potential market application prospects in the related fields of tumor biology, cell transplantation, humanized animal models and the like.
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Inventors:
WEI HONGJIANG (CN)
CHEN QINGFENG (CN)
ZHAO HENG (CN)
JIAO DELING (CN)
ZHAO HONGYE (CN)
CHEN QINGFENG (CN)
ZHAO HENG (CN)
JIAO DELING (CN)
ZHAO HONGYE (CN)
Application Number:
PCT/CN2021/128723
Publication Date:
May 11, 2023
Filing Date:
November 04, 2021
Export Citation:
Assignee:
UNIV YUNNAN AGRICULTURAL (CN)
International Classes:
C12N15/12; A01K67/027; C12N5/077; C12N5/10; C12N15/85
Domestic Patent References:
| WO2018035388A1 | 2018-02-22 |
Foreign References:
| CN106535630A | 2017-03-22 | |||
| CN107119076A | 2017-09-01 |
Other References:
GAO MENGYU, ZHANG BINGQI, HE YUTING, YANG QING, DENG LIHONG, ZHU YUQI, LAI ENJIANG, WANG MENGHUA, WANG LADUONA, YANG GUANG, LIAO G: "Efficient Generation of an Fah/Rag2 Dual-Gene Knockout Porcine Cell Line Using CRISPR/Cas9 and Adenovirus", DNA AND CELL BIOLOGY, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 38, no. 4, 1 April 2019 (2019-04-01), US , pages 314 - 321, XP093062272, ISSN: 1044-5498, DOI: 10.1089/dna.2018.4493
LEI SHAOHUA, JUNGHYUN RYU, KE WEN, ERICA TWITCHELL, TAMMY BUI, ASHWIN RAMESH, MARIAH WEISS, GUOHUA LI, HELEN SAMUEL, SHERRIE CLARK: "Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency", vol. 6, 27 April 2006 (2006-04-27), pages 25222, XP093062274
DATABASE Nucleotide 7 April 2014 (2014-04-07), ANONYMOUS: "Cloning vector pGL3-U6-sgRNA-PG, complete sequence", XP055566618, retrieved from NCBI Database accession no. KJ175229.1
AZUMA, H. ET AL.: "Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg −/− mice", NAT BIOTECHNOL., vol. 25, no. 8,, 31 August 2007 (2007-08-31), pages 903 - 910, XP002493009, DOI: 10.1038/nbt1326
REN JILONG, DAWEI YU, JING WANG, KAI XU, YANAN XU, RENREN SUN, PEIPEI AN, CHONGYANG LI, GUIHAI FENG, YING ZHANG, XIANGPENG DAI, HO: "Generation of immunodeficient pig with hereditary tyrosinemia type 1 and their preliminary application for humanized liver", CELL & BIOSCIENCE, vol. 12, 7 March 2022 (2022-03-07), pages 26, XP093062206
LEI SHAOHUA, JUNGHYUN RYU, KE WEN, ERICA TWITCHELL, TAMMY BUI, ASHWIN RAMESH, MARIAH WEISS, GUOHUA LI, HELEN SAMUEL, SHERRIE CLARK: "Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency", vol. 6, 27 April 2006 (2006-04-27), pages 25222, XP093062274
DATABASE Nucleotide 7 April 2014 (2014-04-07), ANONYMOUS: "Cloning vector pGL3-U6-sgRNA-PG, complete sequence", XP055566618, retrieved from NCBI Database accession no. KJ175229.1
AZUMA, H. ET AL.: "Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg −/− mice", NAT BIOTECHNOL., vol. 25, no. 8,, 31 August 2007 (2007-08-31), pages 903 - 910, XP002493009, DOI: 10.1038/nbt1326
REN JILONG, DAWEI YU, JING WANG, KAI XU, YANAN XU, RENREN SUN, PEIPEI AN, CHONGYANG LI, GUIHAI FENG, YING ZHANG, XIANGPENG DAI, HO: "Generation of immunodeficient pig with hereditary tyrosinemia type 1 and their preliminary application for humanized liver", CELL & BIOSCIENCE, vol. 12, 7 March 2022 (2022-03-07), pages 26, XP093062206
Attorney, Agent or Firm:
BEIJING LONGDA HENGSHENG INTELLECTUAL PROPERTY AGENCY CO., LTD (CN)
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