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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, which is applied in the direction of recombinant DNA technology and the use of vectors to introduce foreign genetic material, etc., can solve the problems of troublesome operation, large BAC, and difficult molecular operation, and achieve the effect of improving efficiency and good effect

Inactive Publication Date: 2013-10-16
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. Double digestion with xho and sal to excise a regulatory element with a Loxp site and eGFP (enhanced Green Fluorescent Protein) eukaryotic expression from the pPB-eGFP-neo plasmid (Wellcome Trust Sanger) The complete sequence was ligated into a PGL3-contro (purchased from PROMEGA, E1741) vector digested with xho to construct a PGL3-loxp-eGFP vector.

[0026] 2. The PGL3-loxp-eGFP plasmid was double-digested with Bgl and Kpn, and the Loxp-eGFP fragment with Bgl and Kpn cohesive ends was cut out and ligated to 7zf-mcs (in PROMEGA's 7zf-mcs) double-digested by 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. The 7zf-loxp-EGFP vector was double-digested with Bgl and not, and the Loxp-eGFP fragment with Bgl and not sticky ends was cut out, and ligated to the plasmid Loxp-F3-neo-F3 double-digested by ...

Embodiment 2

[0029] Example 2 Construction of pCC1BAC-Tol2 vector

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

[0031] 2. The pABRG recombinant plasmid was electroporated into three BACs (numbered 706H15, 169D05 and 572K17) which were electro-competent and identified.

[0032] 3. The above-identified positive clones were prepared into BAC electroshock competent.

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

[0034] 5. Electroshock the recovered PCR fragments into the BAC electroshock competent.

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

[0036] 7. The correctly identified pCC1BAC-Tol2 positive clone was prepared to be electroshock competent for recombination.

[0037] There are 3 different BAC clone numbers in total, and the...

Embodiment 3

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

[0040] 1. The above-identified pCC1BAC-Tol2 positive clone was prepared to be electroshock competent containing the recombinant plasmid pABRG.

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

[0042] 3. The recovered PCR fragment was electroporated into the electroshock competent pCC1BAC-Tol2 containing the recombinant plasmid pABRG prepared above.

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

[0044] 5. Carry out endotoxin-free extraction of firm and correct positive clones, and prepare for electrotransfection.

[0045] Likewise, the vectors were marked as 706H15-Tol2-eGFP, 169D05-Tol2-eGFP and 572K17-Tol2-eGFP vectors, and their sequences were shown in SEQ ID No. 3, SEQ ID No. 8 and 9, respectivel...

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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 utilizing bacterial artificial chromosome homologous recombination. Background technique [0002] Bacterial artificial chromosomes (BACs) 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 of general expression vector genome integration, especially for promoters and regulatory sequences. Clear high-efficiency specific expression genes have more obvious advantages, so BAC is also an ideal high-efficiency specific expression vector for researchers. But the disadvantage is that the BAC is large, the molecular manipulation is difficult, and the transfection efficiency is low. At present, most of BAC is carried out by microinjectio...

Claims

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Application Information

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