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Self-deleting free carrier and application thereof

A carrier and sequence technology, applied in the field of genetic engineering, can solve problems such as affecting transgenic animals, failing to pass animal biosafety assessment, and hindering the commercial application of transgenic pigs

Active Publication Date: 2014-04-16
北京科芙兰德生物科学有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the marker gene mainly has the following disadvantages: first, this marker gene has an effect on the expression of adjacent genes. Second, transgenic animals containing the marker gene cannot pass the animal biosafety assessment, hindering the commercial application of transgenic pigs
[0003] Although the marker gene can also be directly deleted with the cre / loxp system, the deletion of the marker gene will leave the loxp sequence in the genome at the same time, and the randomly inserted loxp sequence is likely to cause insertional mutations of the gene, which will cause potential biological safety hazards to the body. It is difficult to pass the safety assessment of transgenic animals, which limits the production and application of transgenic animals

Method used

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  • Self-deleting free carrier and application thereof
  • Self-deleting free carrier and application thereof
  • Self-deleting free carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Construction of self-deletion vector

[0024] The vector construction steps are as follows, and the construction process is as follows figure 1 ;

[0025] 1. The pEPI-eGFP vector was digested with NheI and AgeI, and the first loxp sequence was inserted to obtain the intermediate vector 1.

[0026] 2. PciI and NheI double digested the intermediate vector 1, deleted the CMV promoter, inserted the human EF1α promoter to replace the CMV promoter, and obtained the intermediate vector 2.

[0027] 3. The intermediate vector 2 was double digested with SalI and MluI, and the large fragment recovered from the gel was linked with the PCR product of SV40PolyA (containing AscI and NotI restriction sites) to obtain the intermediate vector 3.

[0028] 4. Double digest pSP72-mOct4-GFPNEO with AscI and NotI, recover the target fragment from the gel and insert it into the intermediate vector 3 to obtain the final vector pEPI-EF1α-EGFP-Oct4-Cre (such as figure 2 ), the ca...

Embodiment 2

[0029] Example 2 Obtaining of MSTN knockout cells

[0030] 1. Landrace Fetal Fibroblast Establishment

[0031] 1) Take Landrace pigs pregnant for 30 days, kill them, take the oviduct and uterus, bandage the exit, transport them back to the laboratory within 2 hours, take out the fetus from the uterus, wash the fetus with DPBS containing antibiotics, transfer it to the ultra-clean workbench, and use ophthalmic scissors to clean the fetus. Remove the fetal head, limbs, and viscera, and rinse with DPBS.

[0032] 2) Use ophthalmic scissors in a 100mm diameter cell culture dish to cut the remaining part as much as possible.

[0033]3) Add a little serum, cut off the tip of the 1ml gun tip with scissors, leaving a part with a diameter of about 40mm or more, connect the 1ml gun, transfer the tissue pieces to the bottom wall of three T25 cell culture flasks, and use an elbow pipette to remove the tissue The blocks are spread out evenly.

[0034] 4) Turn the side covered with tissue...

Embodiment 3

[0044] Embodiment 3 The embryonic nuclear transfer of MSTN knockout pigs and the preparation of cloned pigs

[0045] 1. In Vitro Maturation of Porcine Oocytes

[0046] Take the ovaries from the slaughterhouse, put them in 28°C-35°C normal saline containing penicillin and streptomycin sulfate, and transport them back to the laboratory within 2 hours. Use a 20mL syringe equipped with a 18-gauge needle to aspirate follicles 3-6mm above the ovary. . Put the extract in a 50mL centrifuge tube, let it stand in a water bath at 37°C for 15 minutes to remove the supernatant, add PVA-TL-HEPES to resuspend the precipitate, let it stand for 15 minutes, repeat once, and put the resuspension into a plastic petri dish with a diameter of 60mm In the process, under a stereomicroscope, use a mouth pipette to select cumulus-oocyte-complexes (Cumulus-Oocyte-complexes, COCs) that are dense and uniform in cytoplasm and are covered with more than 2 layers of cumulus, and washed with mature culture m...

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Abstract

The invention relates to the field of genetic engineering, and particularly provides a self-deleting free carrier. A core function region of the free carrier comprises a human EF1alpha promoter sequence, a 1oxp sequence, an enhanced green fluorescent protein (EGFP) encoding sequence, an S / MAR sequence from a human beta interferon gene, a PolyA sequence of SV40, an expression cassette sequence of a mice Oct4 promoter drive Cre gene, and a 1oxp sequence from 5'-terminal to 3'-terminal. Preparation of gene targeting cells and animals is carried out by using the carrier disclosed by the invention, genes can be deleted, screened and marked and no gene insertion mutation is generated, and the effect on adjacent gene expression caused by a marker gene and potential biological safety hazard on an organism are removed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a self-deleting free vector and its application. Background technique [0002] The physiological characteristics of pigs are similar to those of humans, and their long life cycle is considered to be an ideal animal model. Therefore, the genome modification of pigs can be used to prepare research models similar to human genetic diseases, and can be widely used in the pathological research of human diseases and the development of therapeutic drugs. In addition, pork is also the main source of animal protein for the people of our country, which plays a key role in improving the living standards of the people. With the development of my country's economy and the improvement of people's living standards, the Chinese people's demand for pork is also increasing. According to statistics, China's annual consumption of pork accounts for more than half of the world's total. The expansio...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/877C12N5/10A01K67/027
Inventor 畅飞方锐赵馨李宁张磊汤波王建武
Owner 北京科芙兰德生物科学有限责任公司
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