The shRNA approach was used to achieve a stable knockdown of HMGB1 gene expression (shHMGB1)71 (link). The recombinant lentiviruses carrying shRNA plasmid targeting human HMGB1 gene were purchased from OriGene Technologies (TL316576V, Rockville, MD, USA). A 50 percent confluent Huh7 cell monolayer was infected with the recombinant lentiviruses at an MOI of 20 in the presence of 8 µg/mL of polybrene, which was then incubated at 37 °C overnight. After 24 h, the culture medium was changed to a puromycin-containing selective medium with a final concentration of 1 µg/mL (Sigma-Aldrich, St. Louis, MO).
Recombinant lentiviruses
Manufactured by OriGene
Sourced in United States
Recombinant lentiviruses are a type of lab equipment used for gene delivery. They are viral vectors derived from the lentivirus family, which are capable of efficiently transducing both dividing and non-dividing cells. These lentiviruses can be engineered to carry and deliver specific genetic material into target cells.
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Lab products found in correlation
2 protocols using recombinant lentiviruses
1
HMGB1 Gene Silencing in Huh7 Cells
2
GRK5 manipulation in H9c2 cells
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The H9c2 rat cardiomyoblast cell line was purchased from American Type Culture Collection (Manassas, VA, United States) and cultured as previously described[11 ,16 -18 (link)]. Recombinant lentiviruses encoding for wild-type full-length GRK5 or for empty vector (control) (OriGene Technologies, Rockville, MD, United States) were propagated and purified via CsCl density gradient ultracentrifugation, as described previously[11 ,19 ]. For CRISPR/Cas9-mediated GRK5 gene deletion, a gRNA sequence was custom-synthesized by Sigma-Aldrich (target ID: RN0000391809, target sequence: 5’-GTGGTTTGAATTTATGCGG-3’) and incorporated into a lentiviral vector (Sigma-Aldrich). Along with negative control CRISPR lentiviral particles (CNCV, Cat #CRISPR12V-1EA, Sigma-Aldrich), this lentivirus was also propagated and purified through cesium chloride density gradient ultracentrifugation.
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