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SgRNA for specific recognition of pig KIT gene, coding DNA thereof and kit, and application thereof

A gene editing and DNA molecule technology, applied in the field of genetic engineering, can solve the problems of interfering with the normal KIT protein expression, affecting the normal migration and survival of melanocytes, etc.

Pending Publication Date: 2020-04-07
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, one base G of intron 17 in the mutated copy is replaced by A, which causes the mRNA precursor to not be spliced ​​normally to form normal mRNA, thereby interfering with the expression of normal KIT protein, affecting the normal migration and survival of melanocytes, making large white pigs and Landrace pigs exhibit white hair traits

Method used

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  • SgRNA for specific recognition of pig KIT gene, coding DNA thereof and kit, and application thereof
  • SgRNA for specific recognition of pig KIT gene, coding DNA thereof and kit, and application thereof
  • SgRNA for specific recognition of pig KIT gene, coding DNA thereof and kit, and application thereof

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preparation example Construction

[0050] In some preferred embodiments, the preparation method includes introducing the gene editing element into the pig-derived biological material, which may optionally include pig somatic cells or pig fertilized eggs, and optionally adopt the calcium phosphate method, lipid The gene editing elements are introduced into pig somatic cells by mass transfection, lentiviral transfection or electroporation; optionally, the gene editing elements are introduced into pig fertilized eggs by fertilized egg microinjection. The gene editing element includes the following (a) or (b):

[0051] (a) described sgRNA or the DNA molecule of coding described sgRNA; And Cas9 related element, described Cas9 related element comprises the DNA of coding Cas9, the mRNA of coding Cas9 or Cas9 protein molecule; The DNA of coding Cas9 and the RNA of coding Cas9 can be Selected to contain functional elements independently, examples of said functional elements include but are not limited to promoters, term...

Embodiment 1

[0062] Construction of Cas9 / sgRNA expression vector that specifically recognizes KIT gene target site, sgRNA activity detection targeting vector construction and sgRNA activity detection.

[0063](1) Construction of Cas9 / sgRNA expression vector. First, select a sequence downstream of the coding region of the pig KIT gene as the target sequence, the target sequence is shown in SEQ ID NO.7, design the sequence responsible for recognizing the target fragment region according to the target sequence, and obtain three sgRNA regions responsible for recognizing the target fragment, Named as sgRNA1, sgRNA2 and sgRNA3 respectively, wherein the sgRNA1 sequence is shown in SEQ ID NO.1, the sgRNA2 sequence is shown in SEQ ID NO.2, and the sgRNA3 sequence is shown in SEQ ID NO.3. For the construction steps of the three Cas9 / sgRNA expression vectors, please refer to the instruction manual of the Cas9 / sgRNA Construction Kit (Catalog.No.VK001-01) of Visunlide Company.

[0064] (2) Constructio...

Embodiment 2

[0067] Screening of Large White Pig Fetal Fibroblast Cell Lines Successfully Deleting Redundant Mutant Copy of KIT Gene.

[0068] (1) Cell transfection and monoclonal culture. The day before the transfection, the primary large white pig embryonic fibroblasts were revived to a 6cm plate, and the cells could be transfected when the cells reached 70-80% confluence. The cell transfection method was electrotransfection based on a nuclear transfection apparatus, and the Basic Primary Fibroblasts Nucleofector Kit (Lonza) was used to perform electroporation under the AmaxaNucleofector (Lonza) single-well nuclear transfection system.

[0069] The specific operation process is as follows: a. Collect cells and adjust the number of cells to 5×10 5 ~1×10 6 / tube, centrifuge at 200g for 5min, and remove the culture medium as much as possible. b. Add 100 μL of electrotransfection reagent to resuspend the cells, and add 5 μg of Cas9 / sgRNA2 expression vector plasmid, and slowly add the elec...

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Abstract

The invention provides sgRNA for specific recognition of a pig KIT gene, a coding DNA thereof and a kit, and application thereof, belonging to the technical field of gene engineering. In the sgRNA, anucleotide sequence responsible for identifying a target fragment region is a sequence as shown in SEQ ID NO.1, a sequence as shown in SEQ ID NO.2 or a sequence as shown in SEQ ID NO.3; and the sgRNAenables a CRISPR / Cas9 gene editing system to delete redundant mutation copies of the KIT gene on a pig genome, so the KIT gene can normally express KIT protein with biological activity, and the hair color of a pig is changed.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sgRNA specifically recognizing the pig KIT gene, its coding DNA, a kit and application thereof. Background technique [0002] The KIT gene has been proved to be the main gene regulating the dominant white coat color of Large White pigs. The KIT gene is located in the 1.2 region of the short arm of chromosome 8 (8p12). The normal KIT gene in pigs is a single copy, located between the PDGFRA gene and the KDR gene, with a total length of more than 200kb, consisting of 21 exons, and the entire coding sequence is 2919bp long. The normal single-copy KIT gene shows a black phenotype, and the KIT gene plays a vital role in the normal migration and survival of melanocyte precursors. In addition to the normal KIT gene (KIT1), Large White and Landrace pigs also have Carries multiple full-length copies containing mutations (KIT2, KIT3, ..., KITn; n≥2). Among them, one base G ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N15/877C12N15/12A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/108A01K2267/02C07K14/47C12N15/113C12N15/8509C12N15/8778C12N15/907C12N2800/107C12N2310/20
Inventor 李奎牟玉莲徐奎刘志国樊自尧
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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