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Novel interference vector for increasing animal lean meat percentage and application thereof

A lean meat rate, animal technology, applied in the field of new vector construction, can solve problems such as low transfection efficiency

Inactive Publication Date: 2013-05-01
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

Non-viral vectors such as liposomes, nanoparticles, microemulsions, and polymer micelles have the advantages of safety and non-immunogenicity, but their transfection efficiency is relatively low, making it difficult to efficiently and stably express the target gene carried. The lentivirus (Ieniivirus) vector is a gene therapy vector developed on the basis of HIV-I (human immunodeficiency virus type I).

Method used

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  • Novel interference vector for increasing animal lean meat percentage and application thereof
  • Novel interference vector for increasing animal lean meat percentage and application thereof
  • Novel interference vector for increasing animal lean meat percentage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of lentiviral interference vector

[0028] 1. Materials and Methods

[0029] 1.1 Materials

[0030] DH5 alpha competent cells; T4 DNA ligase (1U / μl) (Invitrogen); pCDNA3.1 (+) vector (Invitrogen Company); EcoR I, Xho I (NEB Company); Ligase (NEB Company). HEK293 cells (Shanghai Invitrogen); DMEM+10% fetal bovine serum (Invitrogen); lipofectamine2000 (Invitrogen); OptiMEM medium (Invitrogen); 0.05% Trypsin (Invitrogen); Trizol (Invitrogen); EagI (NEB); II (Invitrogen); pDONR221 (Invitrogen); pLenti6.3 / V5-DEST (Invitrogen); packaging plasmid (Invitrogen); 293FT cells (Shanghai Invitrogen); 0.05% Trypsin (Invitrogen); DMEM+10% FBS (Invitrogen); Opti-MEM medium (Invitrogen).

[0031] 1.2 Method

[0032] (1) Construction of interference carrier

[0033] Search the GenBank database ( http: / / www.ncbi.nlm.nih.gov / nucleotide ), to obtain the mRNA sequence of sheepmyostatin gene, its sequence number is NM_001009428. According to the Invitrogen miRNA...

Embodiment 2

[0071] Example 2 Application of Lentivirus Interference Vector in Breeding New Breeds of Sheep

[0072] The miRNA lentivirus interference expression vector pLenti6.3-MR085_1 was mixed with trypan blue to prepare a transfection solution. Then, using minimally invasive surgery, the above-mentioned transfection solution was injected into the testes on both sides of the sheep in multiple points, and then mated with females.

[0073] Preliminary research results show that the progeny of the positive transgenic sheep expressing green fluorescent protein was detected by real-time quantitative PCR technology and found that the transcription level of the MSTN gene in the muscle tissue of the sheep was significantly lower than that of the non-transgenic sheep. The results are shown in Table 4.

[0074] Table 4 MSTN gene transcription level

[0075]

[0076]

[0077]

[0078]

[0079]

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Abstract

The invention discloses a novel slow-virus interference vector pLenti6.3-MR085_1 for increasing animal lean meat percentage, of which the sequences comprise gene sequences Sequence NO.1 and Sequence NO.2. In the invention, multiple pairs of miRNA (micro ribose nucleic acid) single-chain oligonucleotides are designed and synthesized for the nucleotide sequence of sheep MSTN gene, an miRNA expression vector is constructed, and a pcDNA<TM>6.2-GW / EmGFPmi-MR085-1 vector with strong inhibitory effect is obtained by screening with functional testing, wherein the vector comprises gene sequences Sequence NO.1 and Sequence NO.2; and the gene sequences Sequence NO.1 and Sequence NO.2 are constructed to the miRNA slow-virus interference vector. The invention also discloses application of the novel vector.

Description

technical field [0001] The invention relates to the construction of a novel carrier. Specifically, the present invention designs and synthesizes miRNA single-stranded oligonucleotides for the nucleotide sequence of the sheep myostatin gene MSTN, constructs miRNA lentiviral interference vectors, and obtains novel vectors with good application prospects. Background technique [0002] People's research on meat traits of livestock and poultry mainly focuses on lean meat percentage and meat quality, and the pursuit of lean meat percentage has always been the focus of efforts of scientists and producers. Muscle yield is an important economic trait of meat livestock and poultry, and plays a decisive role in the economic benefits of animal husbandry. In recent years, with the development of molecular genetics and the innovation of molecular biotechnology, breeders have bred many breeds of livestock and poultry with fast growth, high lean meat rate, and feed saving, and have studied...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/85
Inventor 苗向阳
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI