Method for screening glutamine synthetase-deficient HEK293 cell line
A glutamine and synthetase technology, applied in the biological field, can solve the problems of no screening ability, only 26% positive rate of cell screening, and inability to screen high gene copy number cell clones, etc., to simplify the culture process and have good clinical application prospects and commercial value, and the effect of improving the expression of recombinant protein
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[0087] Example 1: Design GS gene sgRNA and construct CRISP / Cas9 plasmid
[0088] (1) Design the sgRNA sequence of GS gene
[0089] By comprehensively comparing the prediction results of multiple online tools, analyzing the number of mismatches, GC content, out-of-frame score and other conditions, in the third to eighth exon interval of the GS gene coding region ( SEQ ID NO. 1) A total of 81 sgRNA sequences were designed (see Table 1 for specific sequences).
[0090] Table 1 sgRNA sequence of GS gene coding region
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[0094] (2) Construction of pX330-GS-sgRNA (E3#01-E8#13) vector
[0095] The 5'end of the sgRNA sequence in Table 1 was added with a BbsI sticky end, the reverse complementary sequence was designed, and the corresponding primers were synthesized (Table 2). The synthesized sgRNA forward and reverse complementary primers were dissolved in T4 polynucleotide kinase wash solution at a final concentration of 0.5 μM each, and catalyzed by T4 Polynucleotid...
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[0100] Example 2: Construction of pShCMV-EGx-GSE(3-8)-xFP plasmid
[0101] (1) Construct pShCMV-EGx-MCS-xFP plasmid.
[0102] Use plasmid pCMV(PacI)-MCS-IRES-EGFP (plasmid map as Picture 12 (Shown) is the template PCR amplification of Enhanced Green Fluorescent Protein (EGFP) gene fragment. EGFPfrag1 primers are 5'acagATCGATgccaccATGGTGAGCAAGGGCGA G and 5'CTGggatccgaattcAGTGGTTGTCGGGCAGCAG; EGFPfrag2 primers are 5'TCACctcgagGCAAGCTGACCCTGAAGTTC and 5'CTACTGagatctTTACTTGTACAGCTCGTCCATG. The PCR reaction uses KAPAHiFiDNA Polymerase, the annealing temperature is 58℃, and the extension is 15s. The recovered EGFP1 fragment and pShCMV-MCS (MCS sequence is shown in SEQ ID NO. 83, and the plasmid map is shown in Figure 13 Shown) The plasmid pShCMV-EGx-MCS plasmid was constructed after ClaI and BamHI digestion treatment, gel recovery and purification, T4DNA ligase ligation, DH5α competent cell transformation, and a small amount of plasmid DNA preparation and identification. The EGFP2 P...
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[0107] Example 3: Rapid sgRNA efficiency detection
[0108] A total of 16 samples of pX330-GS-sgRNA (E3#01) to (E3#16) and pShCMV-EGx-GSE3-xFP plasmid; pX330-GS-sgRNA (E4#01) to (E4#10) and pShCMV- A total of 10 samples of EG x-GSE4-xFP plasmid (E4#2-1 and E4#2-2 were co-transfected in equal mass ratio); pX330-GS-sgRNA (E5#01) to (E3#15) and pShCMV -EGx-GSE5-xFP plasmids have 15 samples; pX330-GS-sgRNA (E6#01) to (E6#10) and pX330-GS-sgRNA (E7#01) to (E7#16) and pShCMV-EGx- A total of 26 samples of GSE(6&7)-xFP plasmid; 13 samples of pX330-GS-sgRNA (E8#01) to (E8#13) and pShCMV-EGx-GSE8-xFP plasmid were co-transfected by calcium phosphate co-precipitation method HEK293 cells. The pX330 plasmid that does not contain the sgRNA sequence and pShCMV-EGx-GSE3-xFP, pShCMV-EGx-GSE4-xFP, pShCMV-EGx-GSE5-xFP, pShCMV-EGx-GSE(6&7)-xFP, pShCMV-EGx-GSE8 -xFP five plasmids co-transfected as a negative control. The experimental steps are as follows:
[0109] (1) Put HEK293 cells in a 12-well ...
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