Gene editing method for electrotransfected bivalve mollusks

A gene editing, bivalve mollusk technology, applied in genetic engineering, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve problems such as difficult application of shellfish gene editing, achieve low cost, equipment Simple requirements and promising results

Pending Publication Date: 2022-03-11
SHANTOU UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to fill in the gaps in the prior art, the present invention takes the fertilized eggs of bivalve molluscs as the operation object, and uses electrotr

Method used

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  • Gene editing method for electrotransfected bivalve mollusks
  • Gene editing method for electrotransfected bivalve mollusks
  • Gene editing method for electrotransfected bivalve mollusks

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Taking Chlamys sinensis as an example, the experiment of electrotransfection bivalve gene editing was carried out.

[0048] Time: October 2020

[0049] In October 2020, male and female individuals of the golden strain with full gonads were selected from the 8-month-old luxurious scallops cultured in the waters of Nan'ao Island, Shantou City, Guangdong Province; the specific steps are as follows:

[0050] 1. Target gene structure analysis.

[0051] According to the published transcriptome information and unpublished genome information of our research group, we jointly analyzed the gene sequence, and it was confirmed that the gene SRB-like-3 related to carotenoid accumulation has 10 intron regions and 11 exon regions (Such as figure 1 Shown in A), wherein the exon overlaps with the CDS sequence; the myostatin gene MSTN has 2 intron regions and 3 exons in total, of which the No. 3 exon is slightly larger than the CDS region (such as figure 1 shown in B).

[0052] 2. Acco...

Embodiment 2

[0060] Taking Chlamys sinensis as an example, the experiment of electrotransfection bivalve gene editing was carried out.

[0061] Time: April 2021

[0062] In April 2021, from the 8-month-old luxurious scallops cultured in the waters of Nan'ao Island, Shantou City, Guangdong Province, select male and female individuals of the golden strain with full gonads; the specific steps are as follows

[0063] 1. Construction of recombinant plasmids. The sgRNA was designed and synthesized according to Example 1, annealed to form a double strand, and ligated with the pSpCas9(BB)-2A-GFP(PX458) plasmid linearized by BbsI digestion. The schematic diagram of the structure is as follows Figure 6 .

[0064] 2. The steps for obtaining fertilized eggs are the same as in Example 1.

[0065] 3. Electrotransfection operation: Keep the recombinant plasmid concentration ≥ 2 μg / μl, add it to high-density fertilized eggs and mix well, set the electric shock parameters at 250V, 400μs, and transfer 8...

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Abstract

The invention belongs to the field of marine biology application, particularly relates to the field of shellfish molecular genetic breeding, and relates to an electrotransfection bivalve shellfish gene editing method, the concentration of recombinant plasmids is kept to be greater than or equal to 2 mu g/mu l, sgRNA and Cas9 protein are mixed into RNP (sgRNA and Cas9 protein complex) according to the volume ratio of 2: 1, the working concentration of the sgRNA is greater than or equal to 50 ng/mu l, the working concentration of the Cas9 is greater than or equal to 250 ng/mu l, the working concentration of the sgRNA is greater than or equal to 50 ng/mu l, and the working concentration of the Cas9 is greater than or equal to 250 ng/mu l; an electrotransfection method is applied to bivalve mollusk gene editing, the electric pulse condition is that the pulse interval is 1s, the number of pulses is 2, the electrode distance is 4mm, pulse voltage and pulse time are changed, the pulse voltage range is 50V-500V, and the pulse time is 100 microseconds-1ms. The method disclosed by the invention can provide a basis for bivalve gene function verification and gene breeding application, and has the advantages of simplicity in operation, low cost, easiness in popularization and the like.

Description

technical field [0001] The invention belongs to the application field of marine biology, specifically belongs to the field of mollusk molecular genetic breeding, and relates to a gene editing method of electrotransfection bivalve mollusk. Background technique [0002] In the CRISPR / Cas system gene editing introduction method, it is mainly divided into three categories, namely, biological transfection (lentivirus, adenovirus, etc.), chemical transfection (liposome, cationic polymer), physical transfection (electroporation staining, microinjection, gene gun). Compared with the above introduction methods, the biological transfection method is relatively less difficult to operate, but there is a possibility of residual cytotoxicity; the most prominent advantage of the chemical transfection method is that it is easy to operate, does not require complicated technical requirements, and does not have the problem of cytotoxicity. The disadvantage is that the stability is difficult t...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N9/22C12N5/10
CPCC12N15/85C12N15/113C12N9/22C12N5/0601C12N2310/20
Inventor 郑怀平陈颖何成
Owner SHANTOU UNIV
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