A ps transposon system and its mediated gene transfer method
A subsystem and transgenic technology, applied in biochemical equipment and methods, microorganisms, nucleic acid vectors, etc., can solve the problem that DNA transposons are rare in vertebrates, and achieve the effect of improving gene transfer efficiency
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Embodiment I
[0044] Embodiment 1, the construction of transposase expression vector pcDNA3.9-PSase
[0045] 1. Synthesis of transposase PSase vector.
[0046] After the PSase transposase sequence was chemically synthesized, it was cloned into the LB vector to construct the pLB-PSase vector, and the plasmid DNA was detected by 1% agarose gel electrophoresis (eg figure 1 ).
[0047] 2. Construction of transposase eukaryotic expression plasmid and in vitro transcription helper plasmid pcDNA3.9-PSase
[0048] The transposase CDS was excised from the pLB-PSase vector with the restriction enzymes BamHI and XhoI, and the 1296bp CDS sequence was recovered from agarose gel. At the same time, the pcDNA3.9 vector was digested with BamHI and XhoI, and the 3.9kb fragment was recovered by cutting the gel as the vector frame. After linking the transposase CDS and pcDNA3.9 vector frame, transform competent cells Top10, pick a single colony into liquid medium LA for culture, and extract the plasmid for ...
Embodiment II
[0050] Embodiment II, the construction of transposon expression vector
[0051] 1. Construction of transgenic donor plasmid pLB-PS vector
[0052] 1.1 Synthesis of two terminal inverted repeat elements of PS (PS 3' and 5')
[0053] The insertion age of PS transposons in the Tc1 / Marinier family was analyzed for multiple species, and molecular reconstruction was carried out on the basis of phylogenetic comparative studies, and highly conserved 5' and 3' TIRs were identified, which were 28 bp transposons, respectively. to the complementary sequence. Add the restriction endonuclease site to this sequence and send it to Huada Gene Company for synthesis. The sequence is shown in Table 1.
[0054] 1.2 Polymerase Amplification of PS Transposable Elements
[0055] The above chemically synthesized single-stranded nucleotide chain PS5't (including the inverted repeat element at the end of PS5' and an enzyme cleavage site, the enzyme cleavage site is used to insert the target gene) and...
Embodiment III
[0070] Embodiment III, PS transposon mediates target gene expression test at mouse cell level
[0071] 1. Recovery and culture of cryopreserved cells
[0072] The pPS-PGK-NEO and pcDNA3.9-PSase plasmids were extracted with OMEGA endotoxin-free plasmid extraction kit (purchased from OMEGA company), and the final concentration of the products was adjusted to 500ng / ul for cell transfection.
[0073]Take out the cryopreservation tube containing human cervical cancer cells (Hela) from liquid nitrogen (cells stored in our laboratory), and immediately put it into warm water at 37-40°C and shake quickly until the cryopreservation solution is completely thawed; after 1-2min Complete rewarming within 10 minutes; transfer the cell suspension to a sterile centrifuge tube, add 5 mL of culture medium, and blow gently; centrifuge the cell suspension at 800-1000 rpm for 5 min, discard the supernatant; add 1 mL of complete cell pellet to the centrifuge tube containing the cell pellet The medi...
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