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Method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination

An artificial chromosome and homologous recombination technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, etc., can solve problems such as troublesome operation, large BAC, and difficult molecular operation, and achieve good results and improve efficiency.

Inactive Publication Date: 2015-04-15
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the disadvantage is that the BAC is large, the molecular manipulation is difficult, and the efficiency of transfecting cells is low
At present, BAC is mostly carried out by microinjection, which requires single cell injection, is not suitable for mass transfection, and is cumbersome to operate and requires high technical requirements.

Method used

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  • Method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination
  • Method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination
  • Method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1L

[0024] Example 1 Construction of LoxP-eGFP-neo vector

[0025]1. Cut out the pPB-eGFP-neo plasmid (Wellcome Trust Sanger) with a Loxp site and eGFP (enhanced Green Fluorescent Protein, enhanced green fluorescent protein) eukaryotic expression regulatory elements from pPB-eGFP-neo plasmid (Wellcome Trust Sanger) with xho and sal The complete sequence was connected to the PGL3-contro (purchased from PROMEGA, E1741) vector digested with xho to construct a PGL3-loxp-eGFP vector.

[0026] 2. Digest the PGL3-loxp-eGFP plasmid with Bgl and Kpn, cut off the Loxp-eGFP fragment with Bgl and Kpn cohesive ends, and connect it to the 7zf-mcs (in the PROMEGA company) that has been digested with Bgl and Kpn On the basis of the pGEM-7zf vector, the cloning site was changed to obtain the 7zf-mcs vector), and the 7zf-loxp-eGFP vector was constructed.

[0027] 3. Digest the 7zf-loxp-EGFP vector with Bgl and not, cut out the Loxp-eGFP fragment with Bgl and not cohesive ends, and connect it to th...

Embodiment 2

[0029] Construction of embodiment 2pCC1BAC-Tol2 vector

[0030] 1. Three correctly identified BAC clones (numbered 706H15, 169D05, 572K17) were prepared to be electroshock competent.

[0031] 2. Transform the pABRG recombinant plasmid into electric shock-competent three BACs (numbered 706H15, 169D05 and 572K17) and identify them.

[0032] 3. Prepare the above-identified positive clones into BAC electric shock competent.

[0033] 4. Design homology arm primers, PCR amplify the Tol2 transposable element (SEQ ID No.5), and recover the fragments amplified by PCR.

[0034] 5. Electric shock the recovered PCR fragments into BAC electric shock competent.

[0035] 6. Pick a single clone for PCR and sequencing identification, and save the strain after identification is correct.

[0036] 7. Prepare correctly identified pCC1BAC-Tol2 positive clones to be electroshock-competent for recombination.

[0037] There are 3 different BAC clone numbers, and the pCC1BAC-Tol2 vectors are respec...

Embodiment 3

[0039] Example 3 Construction of plasmid pCC1BAC-Tol2-eGFP

[0040] 1. Prepare the correctly identified pCC1BAC-Tol2 positive clones to be electroshock-competent containing the recombinant plasmid pABRG.

[0041] 2. Design homology arm primers, PCR amplify the sequence fragments carrying LoxP-eGFP-neo in the LoxP-eGFP-neo vector, and recover the PCR-amplified fragments.

[0042] 3. Transfer the recovered PCR fragment into the electroshock-competent pCC1BAC-Tol2 prepared above containing the recombinant plasmid pABRG by electric shock.

[0043] 4. Pick positive clones for cross-fragment PCR and sequencing identification, and save the strain after identification is correct.

[0044] 5. The firm and correct positive clones were extracted without endotoxin and prepared for electrotransfection.

[0045] Similarly, the vectors were labeled as 706H15-Tol2-eGFP, 169D05-Tol2-eGFP and 572K17-Tol2-eGFP vectors, and the sequences are shown in SEQ ID No.3, SEQ ID No.8 and 9, respectively...

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Abstract

The invention provides a method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination. Homologous recombination of BAC containing target genes is carried out through use of pABRG recombinant plasmid, then through use of nucleofection technology, the recombined BAC transfects host cells, through adoption Tol2 transposon optimized by mammal codons, so that the recombined BAC containing the target genes is integrated in to host cell genomes. Through successful construction of eGFP marker gene containg human UBC promoter and BAC carrier of eukaryotic screening resistant neo gene, wherein both ends of the carrier being provided with Loxp components, preparation for marking and resistibility removing after cell transfection are ready. In addition, transfection efficiency of electrotransfection BAC-Tol2-eGFP carrier can reach 1%-6%, the highest transfection efficiency can reach 10%, and a complete set of experimental routes and methods for use and operation of large mammalian BAC are provided.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for improving cell transfection efficiency by using bacterial artificial chromosome homologous recombination. Background technique [0002] Bacterial Artificial Chromosomes (BAC) are commonly used to clone DNA fragments of about 150kb-300kb in size. Due to the large size of BAC (up to 300kb), the 5' and 3' regulatory regions are long, and the regulatory elements are complete, which can effectively prevent the position effect after the integration of the general expression vector genome, especially for promoters and regulatory sequences. The clear and efficient specific expression of genes has more obvious advantages, so BAC is also an ideal high-efficiency specific expression vector for researchers. However, the disadvantages are that the BAC is large, the molecular manipulation is difficult, and the efficiency of transfecting cells is low. At present, BAC is mostly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/63
Inventor 曹忠洋阮卫民郑合勋
Owner HENAN UNIVERSITY
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