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Complete set of sgRNA for specifically recognizing pig Wip1 gene as well as application thereof and product

A complete set of genetic technology, which is applied in the field of complete set of sgRNA for specific recognition of pig Wip1 gene and its application and products, can solve problems such as blank research, achieve efficient targeted modification, reduce off-target phenomenon, and strong specificity

Active Publication Date: 2020-03-31
南宁壮博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the role of Wip1 gene in large mammals such as pigs, and the research on whether Wip1 gene plays a role in pig spermatogenesis and motility and related molecular mechanisms is still blank

Method used

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  • Complete set of sgRNA for specifically recognizing pig Wip1 gene as well as application thereof and product
  • Complete set of sgRNA for specifically recognizing pig Wip1 gene as well as application thereof and product
  • Complete set of sgRNA for specifically recognizing pig Wip1 gene as well as application thereof and product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Construction of a complete set of sgRNA expression plasmids targeting the Wip1 gene

[0080] Use the cas9 / gRNA construction kit (Catalog.No.VK001-01) of Beijing Weishang Lide Biotechnology Co., Ltd. to complete the construction. The construction process is as follows:

[0081] (1) According to the two target sequences of the porcine Wip1 gene, the corresponding primer sequences were designed and synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. The specific sequences are shown in Table 1:

[0082] Table 1 Two sgRNA primer sequences

[0083] Nucleotide name Sequence (5'-3') serial number Wip1-sg1-F aaacaccgGTGAGCGTCTTTCTCCGACCA SEQ ID NO.8 Wip1-sg1-R ctctaaaacTGGTCGGAGAAGACGCTCAC SEQ ID NO.9 Wip1-sg2-F aaacaccgGGAGGACGTTACTCAGATCG SEQ ID NO.10 Wip1-sg2-R ctctaaaacCGATCTGAGTAACGTCCTCC SEQ ID NO.11

[0084] (2) Formation of oligonucleotide dimers (oligoduplex)

[0085] Dilute the synthesized olig...

Embodiment 2

[0102] Example 2 Establishment of fragment knockout of Wip1 gene pig fetal fibroblast cell line

[0103] 1. Efficiency verification of sgRNA expression plasmid

[0104] (1) Cell transfection

[0105] The day before the transfection, the primary Meishan pig fetal fibroblasts were resuscitated into a 6cm plate, and the cells could be transfected when the cells reached 70-80% confluence. The transfection step was performed strictly according to the instructions of the Basic Primary Fibroblasts Nucleofector Kit (Lonza). Specifically, 5 μg of each of the recombinant plasmids Cas9 / gRNA-Wip1-sg1 and Cas9 / gRNA-Wip1-sg2 obtained in Example 1 were transfected into porcine fetal fibroblasts by means of electroporation, and the two recombinant plasmids were co-transfected. After transfection, three kinds of recombinant cells were obtained.

[0106] (2) Detection of targeting efficiency

[0107] After the electrotransfected cells were cultured for 48 hours, some of them were used for p...

Embodiment 3

[0117] Example 3 Method for preparing gene-edited pigs with fragment knockout of Wip1 gene by somatic cell nuclear transfer technology

[0118] The cells obtained in Example 2 were used as nuclear transfer donor cells, and young pig oocytes matured in vitro for 40 hours were used as nuclear transfer recipient cells. The nuclear transfer donor cells were transferred into enucleated oocytes, and electrofusion and activation were performed. , to construct recombinant cloned embryos, and select the cloned recombinant embryos with good development status to be surgically transferred into the uterus of natural estrous large white sows for pregnancy. After the embryo transfer, the technicians pay attention to observe the return to estrus, and regularly check the pregnancy of the recipient sows. The piglet ear tissue was collected after birth, and its genome was extracted, and the extracted genome was used as a template to amplify using primers Wip1-F and Wip1-R, and the bands were ob...

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Abstract

The invention, which relates to the technical field of gene engineering, particularly provides a complete set of sgRNA for specifically recognizing a pig Wip1 gene as well as application thereof and aproduct. The sgRNA set comprises Wip1-sgRNA1 and Wip1-sgRNA2 and has the high specificity and the off-target phenomenon can be remarkably reduced, so that the pig Wip1 gene is accurately and efficiently modified in a targeted mode. The sgRNA set specifically recognizes the first exon of the pig Wip1 gene, so that the off-target phenomenon caused by a CRISPR / Cas9 system is effectively reduced andthe adverse effect of non-specific cleavage on a genome is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a set of sgRNA specifically recognizing pig Wip1 gene and its application and product. Background technique [0002] Wild-type p53-induced phosphatase 1 (Wip1) was first discovered in the study of lymphoma, and it was confirmed that it is closely related to wild-type p53; at the same time, through immunofluorescence experiments, The Wip1 protein was found to be localized in the nucleus. Wip1 gene is a new proto-oncogene, which has many functions and plays an important regulatory role in cell growth and development, cell proliferation and migration, DNA damage repair, tumorigenesis, cell homeostasis, disease, aging, etc. . Wipl is a serine / threonine phosphatase. The relative molecular mass of human Wip1 protein is 61KD, with 605 amino acids; the relative molecular mass of mouse Wip1 protein is 66KD, with 598 amino acids. [0003] The expression of Wip1 in mouse embr...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N5/10
CPCC12N9/16C12N15/113C12N15/907C12N2310/20
Inventor 牟玉莲李奎徐奎张秀玲刘志国
Owner 南宁壮博生物科技有限公司
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