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Method for obtaining genetically edited sheep and its special sgRNA and Oligo DNA

A DNA molecule and sheep technology, applied in the field of animal genetic engineering, can solve the problems of FSH concentration differences, incompletely clear mechanism of action, high testosterone level, etc., and achieve the effect of increasing litter size and facilitating cell and individual identification

Active Publication Date: 2019-08-16
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the FecB gene can increase the number of ovulations, its mechanism of action is still not completely clear. Studies have found that FecB gene carriers and non-carriers are completely consistent in phenotype, the length of the estrus cycle is the same, and the LH peak interval from estrus to ovulation is also different. However, there are significant differences in FSH concentration, and the testosterone level of FecB gene carriers is relatively high.

Method used

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  • Method for obtaining genetically edited sheep and its special sgRNA and Oligo DNA
  • Method for obtaining genetically edited sheep and its special sgRNA and Oligo DNA
  • Method for obtaining genetically edited sheep and its special sgRNA and Oligo DNA

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Experimental program
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Effect test

Embodiment 1

[0028] Preparation of sgRNA and Cas9 mRNA

[0029] 1. Design the target sequence of sheep FecB gene and the sgRNA that recognizes the target sequence

[0030] Two sgRNAs are designed to target the target sequence of the sheep FecB gene (the target sequence is on exon 1 of the sheep FecB gene), see sequence 3 and sequence 4 in the sequence listing.

[0031] 2. Preparation of sgRNA FECB

[0032] Send the designed sequence 3 and sequence 4 to Huada Genomics Company, synthesize sgRNA, and dissolve it for use;

[0033] 3. Preparation of Cas9 mRNA

[0034] Send the known Cas9 mRNA sequence to BGI, and synthesize the mRNA for use.

[0035] The Cas9 mRNA sequence is shown in sequence 8 of the sequence listing.

Embodiment 2

[0037] sgRNA / Cas9 mRNA mutation efficiency detection

[0038] 1. Obtaining fertilized sheep eggs

[0039] 1. Oocyte maturation

[0040] Collect sheep ovaries from the slaughterhouse, sterilize and wash them with normal saline for 3-4 times, extract oocytes, wash them with maturation solution for 3-4 times, then add balanced maturation solution into 2 cultured in a 38.6°C incubator (the following incubator cultures are all under the same conditions).

[0041] 2. In vitro fertilization of oocytes

[0042] (1) The oocytes matured in vitro for 24-26 hours were taken out, gently blown with 0.1% hyaluronidase to remove granulosa cells, washed 3 times with fertilization fluid, and then put into balanced fertilization droplets.

[0043](2) Take the frozen semen (the semen comes from Kazakh sheep), thaw it in a water bath, transfer it to the well-balanced fertilized fluid, put it in the incubator for 20-25 minutes, then absorb the upper semen, centrifuge it at 1500rpm for 4-5 minute...

Embodiment 3

[0070] Production of gene-edited sheep

[0071] 1. Selection of experimental sheep: select healthy adult sheep rams and ewes with good production performance and no FecB mutation site as embryo donor sheep; ewes as recipient ewes.

[0072] 2. Simultaneous estrus and superovulation: Donor ewes were treated with CIDR+FSH+PMSG+LH method, the total dose of FSH was 12.2 ml, and intramuscular injections were given in descending order, twice a day with an interval of 12 hours. The first day of placing the CIDR vaginal suppository was set as day 0, intramuscular injection of FSH was started on the 9th to 11th day, and the injections were divided into 7 times, with a total injection dose of 12.2 ml. When the penultimate intramuscular injection of FSH was performed, the CIDR suppository was withdrawn and replaced. Intramuscular injection of PMSG, test estrus 24-48 hours after removal of CIDR, immediate intravenous or intramuscular injection of LH if estrus occurs, vaginal insemination ...

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Abstract

The present invention provides a method for obtaining a genetically edited sheep by RNA-mediated specific-mutation FecB gene and specific sgRNA and Oligo DNA thereof. A system capable of specificallytargeting and modifying a sheep FecB gene includes two sgRNAs and three Oligo DNA sequences, wherein the two sgRNAs are sgRNAFecB-1 and sgRNAFecB-2, which are the RNA shown by the nucleotides of the sequences 3 and 4 of a sequence table or the RNA having the sequence; and the three Oligo DNA sequences are the sequences 5, 6 and 7 of the sequence table or the DNA having the sequence. The sgRNA andOligo DNA provided by the invention have high specificity and can precisely target the sheep FecB gene to realize gene mutation; and facilitates cell and individual identification after gene editing.At the same time, the method can effectively increase the lambing rate.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and relates to CRISPR / Cas9 technology, in particular to a method for obtaining gene-edited sheep through RNA-mediated specific mutation of FecB gene and its special sgRNA and Oligo DNA. Background technique [0002] The reproductive performance of sheep is a quantitative trait of very important economic value to the sheep industry. The selection of lamb size is not only limited by sex and age, but lamb size is a quantitative trait with very low heritability. It is only about 0.1, so it is difficult to improve litter size traits with conventional breeding techniques. FecB (fecudity booroola) is an autosomal mutant gene found in Booroola Merino sheep that can increase the number of ovulations and litters, and is the first high-fecundity major gene identified in sheep . The FecB gene was mapped to the interval corresponding to human chromosome 4q22-23 on sheep chromosome 6, and the BMPR-IB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90
CPCC07K14/47C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 周平李伟周琪王立民曲彬唐红许凯郭延华张译元
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI