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.
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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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