Specific transgenic carrier and construction method thereof

A tissue-specific, transgenic vector technology, applied in the biological field, can solve the problems of incomplete regulatory elements, low expression of downstream protease genes, no visible markers, etc., and achieves the effects of good compatibility, simple identification, and efficient integration efficiency.

Active Publication Date: 2014-01-15
国科润风(广州)生物医药有限责任公司
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Problems solved by technology

Although these two vectors can be used for drug screening, they have no visible markers, and it is difficult to confirm the screening of transgenic cells. They are still traditional vectors, and their integration mode is similar to that of the mouse parotid protein vector constructed by Serguei P. Golovan
However, due to the lack of thorough research on the porcine parotid protein promoter, the current analysis shows that in its upstream 10Kb regulatory region, the regulatory elements are incomplete, and there may be other enhancer regions in other regions, resulting in low expression of the downstream protease gene

Method used

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  • Specific transgenic carrier and construction method thereof
  • Specific transgenic carrier and construction method thereof
  • Specific transgenic carrier and construction method thereof

Examples

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Embodiment 1

[0041] The construction method of the transgene carrier specific for embodiment 1 salivary gland tissue

[0042] see image 3 , is a construction plan diagram of the salivary gland tissue-specific transgene vector of the present invention, comprising the following steps:

[0043] (1) Cloning of mouse parotid protein upstream regulatory region (PSP) (including parotid protein transfection recording start site and first intron)

[0044] The tail tissue samples of the FVB strain mice were taken, and the genomic DNA was extracted, followed by PCR amplification using the FVB mouse genomic DNA as a template, using KOD-FX polymerase (TOYOBO, Japan). First, primers mpsp3497-F1 (SEQ ID NO: 1) and mpsp16390-R1 (SEQ ID NO: 2) were used for the first PCR amplification to obtain the upstream regulatory region of the mouse parotid gland protein (-11503bp~+1390bp). The results are as follows: Figure 4 Shown in A; then using nested primers mPSP-3675-F2 (SEQ ID NO: 3) and mPSP-15980-R2 ...

Embodiment 2

[0093] Example 2 Using the vector constructed in Example 1 to screen transgenic cells

[0094] Using conventional methods, the pPB-MusPSP-neo-EGFP vector constructed in Example 1 was used to transfect porcine fetal fibroblasts, and the results were observed after 14 days of screening with G418. The result is as Figure 13 As shown, the results show that the carrier greatly improves the efficiency of transgenic cell screening under the cooperation of auxiliary enzymes.

[0095] The results of flow cytometry analysis of its screening efficiency are as follows: Figure 14 shown, from Figure 14 It can be seen that as the molar ratio of mPB / PSP increases, the number of green fluorescent cells in groups Z1-Z6 increases first and then decreases. When the molar ratio of mPB / psp reaches 1 / 4, the positive rate of green fluorescent cells is the highest, reaching 22.16%, 1116 positive cell clones, which is 65.65 times of normal transfection linearized plasmid (control 1). When the mP...

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Abstract

The invention discloses a salivary gland tissue specific transgenic carrier and a construction method thereof. The salivary gland tissue specific transgenic carrier is obtained by emerging an upstream regulation region sequence of a mice parotid gland protein promoter and a visual screened neo-EGFP combined double-label to a piggyBac transposon system and a Lox-Cre enzyme system; the transgenic carrier can make protein express in a particular tissue to achieve a high-efficient large-section emerging efficiency. The carrier also has a green luminescence label which is convenient for screening and a neomycin resistance gene which is convenient for eukaryotic cell screening, and makes the screening of transgenic cells and identification of transgenic animals be simpler, more direct, and more accurate. The transgenic carrier provided by the invention is a salivary gland specific expression high-efficient integration universal carrier, and is capable of being applied to fields of transgenic animals such as transgenic mice and pig, and the like. The invention clones the upstream regulation region of FVB mice for the first time, the cloning method is simple and high efficient, and the difficulty in construction of a large section carrier is effectively overcome.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention relates to a salivary gland tissue-specific high-efficiency integrated transgene carrier and its construction method. The invention also relates to a PCR amplification fragment of the mouse parotid gland protein upstream regulatory region and its cloning method . Background technique [0002] Traditional vector construction mainly uses genetic engineering restriction endonuclease, T4 ligase and PCR technology to connect the target fragment to the corresponding vector backbone, and then completes the vector construction after enzyme digestion identification and sequencing verification. According to the needs of the experiment, sometimes multiple fragments need to be ligated, often due to the restriction of the restriction enzyme site, the experimental design becomes more and more difficult, the larger and larger the vector fragments lead to the low efficiency of T4 ligase, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/85
Inventor 张献伟吴珍芳李紫聪刘德武
Owner 国科润风(广州)生物医药有限责任公司
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