The small hairpin (sh)RNA sequence of PCSK9 was identified on the Sigma-Aldrich website (www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/individual-genes.html ) and synthesized by GENEWIZ (Suzhou, China). The recombinant plasmid was added to the Escherichia coli DH5α cells (CWBIO), according to the molecular cloning manual. The mixed liquid was evenly coated on the solid lysogeny broth (LB) medium plate and cultured 12–16 h at 37°C in a humidified incubator. The discrete white colonies were inoculated into 5 ml LB liquid culture medium containing 4-(aminomethyl) piperidine (100 g/ml) then placed in a constant temperature oscillator overnight. The plasmid was extracted using the PurePlasmid Mini kit (CWBIO; Beijing, China). The lentiviral packaging system containing three helper plasmids (Rev 2.5 µg, VSVG 3 µg and pMDL 5 µg) and 279-target plasmid (279-vector 12 µg, 279-iPCSK9-1 12 µg and 279-iPCSK9-2 12 µg) were co-transfected into 293T cells using polyethylenimine. The virus was collected and transfected into EAhy926 cells. shRNA-PCSK9 sequences are presented in Table I .
Escherichia coli dh5α cells
Manufactured by CWBIO
Escherichia coli DH5α cells are a well-characterized strain of E. coli bacteria commonly used in molecular biology research. These cells are designed for efficient DNA cloning and plasmid amplification, with high transformation efficiency and stable maintenance of plasmids.
Automatically generated - may contain errors
2 protocols using escherichia coli dh5α cells
1
Lentiviral Knockdown of PCSK9 in EAhy926 Cells
2
Bisulfite Sequencing of Trpm1 Promoter
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Our WGBS performed on the hippocampus of male offspring from ECC (date not shown) revealed a differentially methylated region (DMR) in the promoter of Trpm1 (Chr7: 64,226,560–64,226,639). Bisulfite sequencing was conducted to observe CpG methylation of this region. Genomic DNA was isolated from hippocampus and other organs using MiniBEST Universal Genomic DNA Extraction Kit (Takara). Bisulfite conversion of DNA was performed using an EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer’s instructions. Bisulfite conversion of DNA from blastocysts was performed as previously described (Chen et al., 2004 (link)). The converted DNA was then amplified by nested PCR (first: forward TTA TTA AAG AAA GTT TTT TGG GGG; reverse TTC TTA CCT TTA ACT AAA AAT TCA TCATC. Second: forward TTA TTT AGT GGG ATA AAT TAT TTA AAT AG; reverse ACA ACA AAT AAT TCT AAA AAC TTC). The PCR products were purified using Gel Extraction kit (CWBiotech), and the purified PCR products were cloned into the pMD19-T Vector (Takara) and transformed into Escherichia coli DH5α cells (CWBiotech). At least 15 clones for each sample were sequenced using an ABI 3730xl sequencing platform (BioSune).
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