Method for editing swine BMP15 (bone morphogenetic protein 15) gene by using CRISPR/Cas9

A gRNA-e1s1, gene technology, applied in the field of editing pig BMP15 gene using CRISPR/Cas9, can solve the problems of loss of reproductive ability of sheep, few research reports, and increase of sheep litter size.

Inactive Publication Date: 2018-11-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BMP15 has been identified as a major gene controlling ovulation and multiple births in sheep, but there are relatively few reports on the effects of BMP15 on the reproductive traits of other livestock such as pigs and cattle
[0003] The role of BMP15 protein in different species is different. BMP15 biallelic knockout mice still have the ability to give birth, but BMP15 biallelic mutant sheep lose reproductive ability, while monoallelic mutant sheep have a lower litter size. Increase

Method used

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  • Method for editing swine BMP15 (bone morphogenetic protein 15) gene by using CRISPR/Cas9
  • Method for editing swine BMP15 (bone morphogenetic protein 15) gene by using CRISPR/Cas9
  • Method for editing swine BMP15 (bone morphogenetic protein 15) gene by using CRISPR/Cas9

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Using CRISPR / Cas9 to Edit Porcine BMP15 Gene to Prepare Related Gene Edited Cells

[0035] 1. Obtaining isolated porcine fetal kidney cells

[0036] Pig fetal kidney cells are isolated from large white pig fetal kidneys, and the pig fetal kidney cells are separated in an ultra-clean bench. Use scissors and tweezers to remove the kidney tissue of the fetus, wash the removed tissue repeatedly in 75% alcohol and PBS with antibiotics in sequence, cut the tissue piece to a size of 1 cubic millimeter with small scissors, and centrifuge at 1600rpm for 5 minutes to remove the PBS. Then add 20% FBS DMEM with antibiotics, gently pipette evenly, and place in a 37°C cell culture incubator for cultivation. After putting it into the cell culture box, do not move the culture dish. Three days later, it can be observed that the pig fetal kidney cells have covered the entire culture dish, and then the digestion and culture of ordinary passaged cells can be carried out.

[0037] 2. Obta...

Embodiment 2

[0051] Example 2: Construction of BMP15 gene-edited pigs using somatic cell nuclear transfer technology

[0052] 1. Somatic cell nuclear transfer to obtain BMP15 gene-edited pigs

[0053] Select ovaries with appropriate developmental stages from healthy Large White sows, extract the contents of follicles with a diameter of 3-5 mm on the surface of the ovary with a syringe, dilute the contents in TL-PVA and resuspend to form a suspension. The suspension was left standing at 37°C until the oocytes were completely precipitated, and the oocytes were sucked out and placed under a stereoscope with a pipette or a suction pipette to pick out oocytes with complete pericytes. The selected healthy oocytes were put into TCM-199 containing 10% follicular fluid, FSH, LH, EGF and cultured for 22 hours. Then use a pipette or mouth pipette to move the oocytes to TCM-199 containing 10% follicular fluid and EGF and continue to culture for 22 hours. After 44 hours of culture and maturation, the...

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Abstract

The invention discloses a method for editing a swine BMP15 (bone morphogenetic protein 15) gene by using CRISPR/Cas9. The method comprises the steps as follows: designing two gRNAs on an exon 1 of theBMP15 of a target swine genome, and constructing pX458 and pX459 vectors respectively, so that the BMP15 gene loses functions due to accurate deletion of DNA fragments. Compared with single gRNA medicated editing, the method has the advantage that the BMP15 gene can lose functions more effectively due to accurate deletion of DNA fragments of the exon.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for editing pig BMP15 gene by using CRISPR / Cas9. Background technique [0002] Bone Morphogenetic Protein 15 (BMP15) gene is specifically expressed in the oocytes of mice, rats and humans, and has the function of promoting the division and proliferation of granulosa cells, and is very important for early follicular development. It is a key gene affecting ovulation rate and litter size in mammals. BMP15 has been identified as a major gene controlling ovulation and multiple births in sheep, but there are relatively few reports on the effects of BMP15 on the reproductive traits of other livestock such as pigs and cattle. [0003] The role of BMP15 protein in different species is different. BMP15 biallelic knockout mice still have the ability to give birth, but BMP15 biallelic mutant sheep lose reproductive ability, while monoallelic mutant sheep have a lower litter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/113C12N15/85
CPCC07K14/51C12N15/113C12N15/8509C12N15/907C12N2310/10C12N2310/20
Inventor 何祖勇陈瑶生刘小红莫德林丛佩清石翾唐涛刘小凤
Owner SUN YAT SEN UNIV
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