Transgene report pig detecting Cre site special recombinase active and culturing method

A culture method and recombinase technology, applied in the field of zoology, can solve problems such as large differences in physiological characteristics and inability to replicate human diseases well, and achieve the effect of improving efficiency

Inactive Publication Date: 2009-10-07
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To monitor the activity of Cre recombinase in vivo, transgenic reporter animals need to be established. At present, due to the immature technology of pig nuclear transfer, transgenic mice are the main models of human diseases, but the hemodynamics, tissue size and corresponding physiological characteristics of mice are different from those of humans. The difference is large, and human diseases cannot be well replicated. The reporter animals established to monitor Cre recombinase activity in vivo are only limited to mice, and no other transgenic reporter animals have been reported.

Method used

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  • Transgene report pig detecting Cre site special recombinase active and culturing method
  • Transgene report pig detecting Cre site special recombinase active and culturing method
  • Transgene report pig detecting Cre site special recombinase active and culturing method

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Experimental program
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Effect test

Embodiment 1

[0058] Insulator, CMV promoter, amplification of green fluorescent protein gene

[0059] Using PCI-Neo as a template, add primers C1 and C2 to amplify the CMV promoter through PCR cycle, the full-length sequence is 1022bp

[0060] Using PBC-1 as a template, add primers I1 and I2 to amplify the insulator through PCR cycle, the full length is 2450bp

[0061] Using pEGFP-C1 as a template, adding primers E1 and E2 to amplify the eGFP gene through PCR cycles, the full-length sequence is 804bp.

[0062] The amplified sequence was connected to PMD-18T-Simple (takara company), and entrusted to Beijing Tiangen Company to perform sequencing. After digesting the sequenced correct fragments, they were respectively connected into the STOP-eGFP-ROSA26TV vector and identified by enzyme digestion After the sequencing was correct, it was named pICE-STOP. After expanding the culture, the plasmid was extracted using Tiangen’s large-scale plasmid extraction kit, and linearized with NheI single-e...

Embodiment 2

[0064] Cell transfection and selection

[0065] Transfection of embryonic fibroblasts using liposome-mediated DNA, steps. Follow the instructions of lipofectin2000 (Invitrogen). The day before the transfection, the primary cultured embryonic fibroblasts were resuscitated into a 60mm plate with antibiotic-free culture medium, and the cells could be transfected when the cells reached 70-80% confluency. 24 hours after transfection, cells were passaged by trypsinization 1:21. 48 hours after transfection, G418 selection medium (concentration: 300 μg / ml) was added. When a single colony of cells appears, remove the culture medium, wash once with PBS at 39°C, put a self-made glass cell cloning ring on the cell colony, add an appropriate amount of trypsin to the cloning ring, and digest for about 1 minute , adding selection medium to terminate the reaction. Carefully blow the cells with a pipette and transfer to a 24-well plate for expanded culture. After the cells are overgrown, t...

Embodiment 3

[0067] Identification at the Cellular Level -

[0068] 1. PCR detection

[0069] The obtained neo gene-positive cell genome was used as a template, and primers C1 and E2 were used to amplify, and the amplified sequence included CMV promoter, Loxp-neo-stop-Loxp sequence, and EGFP gene. It proves that the target gene is fully integrated into the genome of porcine embryonic fibroblasts.

[0070] 2. Fluorescence observation

[0071] In order to verify whether the constructed reporter cell line conditionally expressing green fluorescent protein can effectively monitor the activity of Cre recombinase, the screened cell clones were expanded and cultured, and then infected with adenovirus expressing Cre recombinase to verify the activity of Cre recombinase. Validation of construction of reporter cell lines.

[0072] 3. RT-PCR

[0073] Three days after the obtained cell clones were infected with the adenovirus, the total RNA of the cells was extracted for RT-PCR reaction to further...

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Abstract

The present invention provides a transgene report pig detecting Cre site special recombinase active and a culturing methodThe method includes steps as follows: building LoxP site anchoring end terminal, receiving a target carrier of report gene, when Cre recombinase exist, deleting the end terminal anchored by Loxp, and expressing the received report gene, accordingly, monitoring Cre recombinase activity; filter out a fibroblast which can be special detected Cre activity through cell transfection and cell filtering technique; using body nucleus transplantation technique for processing cloning pig. The method is used for monitoring Cre recombinase activity in body, provides a monitoring tool for establishing human disease model, solves problems of immature technique for transgene cloning pig and low efficiency.

Description

Technical field: [0001] The invention provides a transgenic reporter pig for detecting the activity of Cre site-specific recombinase, and also discloses its cultivation method, which is used to monitor the activity of Cre recombinase in vivo, provides a monitoring tool for establishing human disease models, and belongs to animals field of technology. Background technique: [0002] Pigs are the most suitable animals for human organ transplantation and the establishment of human disease models. Establishment of animal models of human disease generally requires Cre recombinase to be expressed in a specific pattern, such as using a tissue-specific promoter. To monitor the activity of Cre recombinase in vivo, transgenic reporter animals need to be established. At present, due to the immature technology of pig nuclear transfer, transgenic mice are the main models of human diseases, but the hemodynamics, tissue size and corresponding physiological characteristics of mice are diffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/06A01K67/027C12Q1/25
Inventor 欧阳红生逄大欣李莉王铁东陈立梅赖良学任林柱张明军李占军
Owner JILIN UNIV
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