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General type gene targeting vector

A gene-targeting, general-purpose technology, applied in the field of bioengineering, can solve the problems of affecting the growth and development of transgenic individuals, reducing developmental potential, and difficult somatic cell nuclear transfer, etc., achieving important scientific value and application prospects

Active Publication Date: 2015-05-27
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, selectable markers such as neo will remain after screening, which may affect the growth and development of transgenic individuals
Second, after positive and negative screening, the primary somatic cells of domestic animals have been treated with drugs twice, and their developmental potential has been greatly reduced, making it difficult for them to be used for somatic cell nuclear transfer.

Method used

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  • General type gene targeting vector
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction and Enzyme Digestion Verification of Universal Gene Targeting Vector

[0026] (1) Sources of main reagents and materials

[0027] pBCP + The vector was purchased from Addgene, and pEGFP-C1-EGFP was purchased from Clontech. Lamda DNA marker and Asel restriction endonuclease were purchased from Fermentas. High-fidelity DNA polymerase KOD Plus and its matching buffer (10×buffer), dNTP, MgSO 4 All were purchased from Toyobo Biotechnology Co., Ltd. The recombinant vector was carried out in accordance with the instructions of the small-scale ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd. DNA tapping gel recovery kit was purchased from Qiagen. Sequencing was entrusted to Shenzhen Huada Gene Co., Ltd. to complete.

[0028]The primers for amplifying the attB sequence are AttB-F:gtc attaat CGCCATTCAGGCTGCGCA (SEQ.ID.NO.2), AttB-R:gtc attaat CTCGGCCTCGACTCTAG (SEQ. ID. NO. 3). The underlined sequence indicate...

Embodiment 2

[0056] Example 2 Screening of Cell Clones Integrated with Universal Gene Targeting Vectors

[0057] (1) Sources of main reagents and materials

[0058] The porcine kidney PK15 cell line was purchased from ATCC. Fugene HD transfection reagent was purchased from Roche. The фC31 integrase expression vector pCMV-фC31 was purchased from Addgene. The preparation of endotoxin-free plasmids was carried out according to the instructions of the small-scale ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd. G418 antibiotic was purchased from Sigma. DMEM, DPBS, fetal bovine serum, Opti-MEM, DMSO were purchased from Invitrogen.

[0059] (2) Operation steps

[0060] - cell plating

[0061] After the PK15 cells were digested with trypsin to form a single cell suspension, according to 10 4 Cells / well were plated in 24-well plates and grown overnight. Before transfection, the cells were washed twice with DPBS pre-warmed to 37°C, and then replaced with...

Embodiment 3

[0070] Qualitative detection of embodiment 3 marker gene deletion reaction

[0071] (1) Sources of main reagents and materials

[0072] In this study, Takara's LA Taq DNA polymerase was used for detection. The corresponding detection primers were M5F and M3R, and the expected product length was about 400bp.

[0073] (2) Operation steps

[0074] LA Taq DNA polymerase PCR

[0075] 1) System:

[0076]

[0077] 2) Cycle condition: (30 cycles)

[0078]

[0079] 3) Electrophoresis:

[0080] ① Electrophoresis of PCR products on 1% agarose gel, sample: 5 μl; 6×Buffer: 1 μl; marker: 2 μl;

[0081] ② Electrophoresis results using ChemiDOC TM XRS+ (Bio-Rad) imaging, observe the results, and take pictures.

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Abstract

The invention discloses a general type gene targeting vector, and belongs to the field of bioengineering. The general type gene targeting vector uses neomycin phosphotransferase and EGFP as positive selective markers and DsRed as a negative selective marker; two sides of a positive selective marker gene expression frame respectively contain LoxP sites which are arranged in a same direction; and the LoxP sites respectively contain multiple cloning sites which include rare restriction enzyme sites nearby, so that the convenience for the cloning of a gene targeting arm is achieved. The general type gene targeting vector contains two homing restriction enzyme sites, and the two homing restriction enzyme sites are respectively I-CeuI and I-SceI and can be used as general endonuclease sites for linearizing the vector. The general type gene targeting vector provided by the invention not only has the functions of single drug and double fluorescent screening, but also provides a new material for an animal safety transgenic technology because the contained LoxP sites are capable of mediating the high-efficiency deletion reaction of the selective markers, and has significant application prospects.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a universal gene targeting carrier. Background technique [0002] Animal transgenic technology has important application value in the analysis of gene functions and signaling pathways, the construction of animal models of diseases, the creation of bioreactors, and the cultivation of new breeds of livestock. Animal transgenic technology can be classified from three levels: molecular means, transfer mode and cell medium. Molecular means mainly refer to the specificity of modification sites, including non-site-specific and site-specific. DNA random integration, virus-mediated, transposase / transposase, etc. are non-site-specific, and their common defects are the inability to strictly control the integration site and copy number, and the expression of foreign genes is also difficult to predict, so it is difficult to apply to transgenic livestock Breeding up. In this contex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
Inventor 毕延震郑新民华再东任红艳张立苹刘西梅华文君李莉肖红卫魏庆信
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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