Skeletal muscle specific promoter and its application
A skeletal muscle-specific promoter technology, which is applied in genetic engineering applications and biological fields, can solve the problem of no relevant reports on the role of miRNA, and achieve high specificity, reduced difficulty, and high activity.
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Embodiment 1
[0021] Construction of pGL3-2eMCK-pMCK-T208-mCherry-EGFP vector:
[0022] 1) Construction of pGL3-mCherry-EGFP vector
[0023] A. Design primers to amplify EGFP, SV40poly(A) and mCherry sequences according to the pCMV-C-EGFP, pGL3-Control and pBABE-puro mCherry-EGFP-LC3B vector sequences, respectively. See Table 1 for primers.
[0024] Table 1 EGFP, SV40poly(A) and mCherry primers
[0025]
[0026] Note in Table 1: The underlined part is the added restriction site, and the bold part is the protected base.
[0027] Table 2 PCR system
[0028]
[0029] PCR reaction procedure: pre-denaturation at 98°C for 3min, denaturation at 98°C for 10s, annealing at 60°C for 10s, extension at 72°C for 2s, denaturation to extension for 35 cycles, and extension at 72°C for 3min.
[0030] Purification and recovery of PCR products: The amplified PCR products were detected by 1% agarose gel electrophoresis, after 20 minutes of electrophoresis, the target band was cut off under ultraviole...
Embodiment 3
[0096] Identification of promoter specificity
[0097] intraperitoneal injection of mice
[0098](1) Dilute 120 μg of plasmid (pGL3-2eMCK-pMCK-mCherry-EGFP or pGL3-2eMCK-pMCK-T2 08-mCherry-EGFP) in 300 μl of 5% sterile glucose solution, shake and mix.
[0099] (2) Dilute, 18 μl HiGene in 300 μl 5% glucose, shake and mix.
[0100] (3) Add the diluted HiGene to the plasmid solution, shake and mix immediately, and centrifuge briefly at low speed to shake the liquid to the bottom of the tube.
[0101] (4) Stand at room temperature for 20 minutes.
[0102] (5) intraperitoneal injection into mice.
[0103] (6) After 48 hours, the myocardial and leg muscles of the mice were collected and stored in liquid nitrogen.
[0104] Tissue-like RNA extraction
[0105] RNA was extracted from heart and muscle tissue by Trizol method, and cDNA was synthesized by reverse transcription kit from TAKARA company
[0106] RNA level detection of promoter specificity.
[0107] According to the mCh...
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