Precision and high-efficiency transgenic carrier as well as construction method and application thereof

A transgenic vector and gene technology, applied in the field of accurate and efficient transgenic vector and its construction, can solve the problem of lack of relevant literature reports, and achieve the effects of reducing subsequent hybridization, improving efficiency and shortening time.

Active Publication Date: 2018-03-06
山东菌胜生物科技有限责任公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

This technology can reduce a lot of experimental steps and save a lot of manpower and material resources. At present, there are many gene knockouts using MCR technology, but there is no relevant literature report on using this technology to transgene

Method used

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  • Precision and high-efficiency transgenic carrier as well as construction method and application thereof
  • Precision and high-efficiency transgenic carrier as well as construction method and application thereof
  • Precision and high-efficiency transgenic carrier as well as construction method and application thereof

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Experimental program
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Embodiment 1

[0041] The overall experimental route of this embodiment is as follows: figure 1 Shown:

[0042] 1. gRNA acquisition:

[0043] The BmFIBH gene sequence of silkworm was retrieved from NCBI (GenBank: AF226688.1). Using the Cas9-gRNA target site prediction web version (http: / / crispr.dbcls.jp / ) that was integrated into the silkworm genome in 2015, candidate gRNA target sites were designed within the 400 bp DNA sequence upstream and downstream of the gene. Candidate target site information (Table 1).

[0044] Table 1 Target design of upstream and downstream of BmFIBH gene

[0045]

[0046] 2. Skeleton carrier acquisition:

[0047] Using the pMD19-T vector as a template, the backbone vector was amplified with high-fidelity Pfsion DNA polymerase. The amplified linearized backbone vector was purified by tapping gel, the circular template plasmid was removed, and stored at -20°C for future use.

[0048] 3. Homology arm acquisition:

[0049] Homology arms are selected as close a...

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Abstract

The invention discloses a precision and high-efficiency transgenic carrier as well as a construction method and application thereof. The transgenic carrier is obtained by completely assembling a genecoding CRISPR/Cas9 proteins, a DNA template of gRNAs, a homologous arm of a target gene side wing and a target gene to be transferred into a same plasmid vector. By adopting the precision and high-efficiency transgenic carrier as well as the construction method and application thereof, the workload and the subsequent hybridization for operating the gene in a laboratory can be significantly reduced, the homozygote generation time can be shortened, and the transgenic efficiency can be significantly increased. Based on an MCR method, the external target gene and the Cas9 gene are jointly transferred into silkworm genome in the process for precisely modifying the gene by virtue of the catalytic effect of the Cas9, the homozygote can be generated in a G0 generation, i.e. the mutation generatedon one copy can be automatically propagated onto an allele.

Description

technical field [0001] The invention belongs to the field of transgenic technology, and in particular relates to a precise and efficient transgenic carrier and its construction method and application. Background technique [0002] Genome editing is an important means of reverse genetics research. It uses various technical means to purposely and precisely modify the genetic information of living cells or organisms, change the expression intensity of genes, and determine the impact of changes at the gene level on organisms. important means of direct influence on somatic phenotypic traits. In the study of gene function, it has experienced early gene overexpression, RNA interference-mediated gene knockout, homologous recombination technology with extremely low targeting efficiency, and meganuclease, zinc finger protease, transcription activator-like effector nucleic acid After the enzyme-mediated knockout of genes, a brand-new gene modification technology-clustered regularly in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90
Inventor 王文刘力源相辉赵若苹张业胜
Owner 山东菌胜生物科技有限责任公司
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