Promoter pCALM2 and application thereof
A promoter, paav-pcalm2- technology, applied to the promoter pCALM2 and its application fields, can solve the problems such as the inability to meet the specificity requirements, and achieve the effects of high labeling efficiency, improved expression specificity, and wide application prospects.
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Embodiment 1
[0036] Example 1 Construction of pUC57-pCALM2 vector
[0037] The pCALM2 promoter is a partial sequence of the calmodulin 2 (CALM2) gene, and the sequence is selected from the partial sequence near the transcription initiation site of the CALM2 gene and the partial sequence of the first exon, totaling 3020 sequences. The nucleotide sequence of the promoter pCALM2 is shown in SEQ ID NO:1.
[0038] The promoter pCALM2 was artificially synthesized and then cloned into the pUC-57 vector to obtain the pUC57-pCALM2 vector.
Embodiment 2
[0039] Example 2 Construction of recombinant adeno-associated virus vector pAAV-pCALM2-eYFP
[0040] In this example, because eYFP is used as the reporter gene of the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the eYFP gene is selected as the vector backbone to connect the pCALM2 promoter, and the steps are as follows:
[0041] like figure 1As shown, the pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and KpnI-HF, while the pUC57-pCALM2 vector was treated with restriction enzymes MluI-HF and KpnI-HF, and digested at 37°C for 3 h , the enzyme digestion system is shown in Table 1 and Table 2, after the recovery of the enzyme digestion product, the linking premix (2×ligation premix, TAKARA) was used for ligation at 16 ° C for 30min, the system was shown in Table 3, and the successfully connected pAAV was obtained - hSyn-eYFP vector.
[0042] Table 1 pAAV-hSyn-eYFP vector digestion syst...
Embodiment 3
[0048] Example 3 Preparation of recombinant adeno-associated virus AAV-retro-pCALM2-eYFP
[0049] In this example, the three-plasmid co-transfection method was used to prepare the virus. Before virus preparation, the plasmids needed to package the virus need to be extracted, including the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP, the packaging plasmid AAV-retro-RCB and the helper plasmid. Specific steps are as follows:
[0050] Preparation of cells: 293T cells were plated in culture dishes containing complete medium (10% fetal bovine serum, 1% double antibody), and cultured in an incubator.
[0051] Prepare transfection reagent: Pipette 5.25mL of ultrapure water, 75μg of packaging plasmid, 75μg of recombinant plasmid, 75μg of helper plasmid and 800μL of calcium chloride solution, and mix gently. To the aforementioned reagents, an equal volume of 2×HBS was added, vortexed and left to stand for 30 min.
[0052] Transfected cells: Add 20 μL of chloroquine to ...
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