Method for preparing porcine kidney epithelial cell line for stably expressing BE4 protein based on piggyBac transposon system

A porcine kidney epithelial cell, stable expression technology, applied in the field of genetic engineering and cell engineering, can solve the problems of affecting gene editing efficiency, low positive rate, low transfection efficiency, etc.

Inactive Publication Date: 2021-01-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since the gene encoding the BE4 fusion protein is relatively large, the transfection efficiency will be low due to the large plasmid when performing...

Method used

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  • Method for preparing porcine kidney epithelial cell line for stably expressing BE4 protein based on piggyBac transposon system
  • Method for preparing porcine kidney epithelial cell line for stably expressing BE4 protein based on piggyBac transposon system
  • Method for preparing porcine kidney epithelial cell line for stably expressing BE4 protein based on piggyBac transposon system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 piggyBac transposon and transposase expression vector construction

[0035] See Figure 1 with image 3 , piggyBac transposon carrier (PB-BE4max) includes elements such as BE4 fusion protein sequence, antibiotic selection marker gene (puromycin resistance gene), 5'TR and 3'TR homologous sequence, the sequence is as SEQ ID NO.1 As shown, the plasmid map as Figure 1B shown. The transposase carrier plasmid includes elements such as the transposase protein sequence, the sequence is shown in SEQ ID NO.2, and the plasmid map is shown in image 3 shown. The plasmid was positively transformed into Escherichia coli, expanded and cultivated, and then extracted by removing endotoxin to obtain the plasmid required for the experiment.

Embodiment 2

[0036] Example 2 PB-BE4max vector and transposase expression vector plasmid co-transfect PK-15 cells

[0037] The above-mentioned PB-BE4max vector and transposase expression vector plasmid were co-transfected into PK-15 cells, and the liposome transfection method (lipo2000) was used. The specific operation process was as follows:

[0038] Before transfection, the well-passaged PK-15 cells were inoculated into 6-well culture dishes, and transfected when the cells grew to a confluence of 70%-80%. According to the ratio of mass ratio PB-BE4max carrier: transposase expression vector = 2:1, the PB-BE4max vector and transposase expression vector were co-transfected into PK-15 cells, and operated according to the operation manual of lipo2000 (Invitrogen) . After the transfected cells were cultured for 48 hours, the medium was replaced with a fresh medium of 2 μg / mL puromycin, and the culture medium was continued at 37°C, 5% CO 2 Cultivate in a constant temperature cell incubator fo...

Embodiment 3

[0039]The picking of embodiment 3 monoclonal cell lines

[0040] refer to Figure 4 , dilute the cells of the positive cell population described in Example 2 to 7-8 cells / mL with fresh basal medium, then plate them in a 10cm dish, and cultivate them for 8-10 days; after the single cells grow into a single cell mass, Use the cloning ring to pick out the positive cell clusters and continue to expand the culture. Figure 4 Shown is a diagram of a monoclonal cell line formed from a single cell.

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Abstract

The invention discloses a method for preparing a porcine kidney epithelial cell line for stably expressing BE4 protein, which comprises the following steps: by using porcine kidney epithelial cells (PK15) with good passage as host cells, co-transfecting a piggyBac transposon (PB-BE4max) vector containing a BE4 fusion protein sequence and a transposase expression vector plasmid, screening a positive cell population by puromycin, and selecting a monoclonal cell line; and verifying the editing activity amplifying the characteristic fragment of BE4 and transfecting active sgRNA, so as to obtain the transformant for stably expressing BE4 protein. The porcine kidney epithelial cell line prepared by the method can stably express BE4 protein, and has important application value in the porcine genetic engineering fields of porcine genome function research, transgenic pig preparation, drug-resistant target screening and the like.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and cell engineering, and in particular relates to a method for preparing a pig kidney epithelial cell line stably expressing BE4 protein based on a piggyBac transposon system. Background technique [0002] As the hottest gene editing technology in recent years, the CRISPR / Cas9 system has been widely used in human basic medical research, animal and plant life sciences and other fields. However, for the correction of genes with single base mutations, the CRISPR / Cas9 system is not widely used because of its low homologous recombination efficiency in cells. When DNA double-strand breaks, cells are more inclined to use the principle of non-homologous end repair to repair the target DNA, so the replacement process of a single base by homologous recombination is very inefficient. David R. Liu and his research team reported a new type of gene editing tool in 2016, that is, base editing (Base Editing,...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N5/10C12R1/91
CPCC12N15/85C12N15/65C12N5/0686C12N2800/107C12N2800/90C12N2510/00
Inventor 赵书红熊友才阮进学韩晓松庄荣志赵广兴李金虎赵长志李长春谢胜松李新云
Owner HUAZHONG AGRI UNIV
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