To generate cells with ABCC5 knockdown and overexpression, lentiviral vectors were amplified with the insert (full-length human ABCC5; NR_135125.2) by qRT-PCR, compared to human reference cDNA. Lentiviruses were produced by transient transfection of 293T cells with pSPAX2, pMD2G, and pHB-U6-MCS-zsgreen-puro (empty) (Hanbio, Shanghai, China) plasmid DNA (2439 -BamHI and 2456 -EcoRI sites) plus LipoFiter (Hanbio Biotechnology, ShangHai, China), following the manufacturer's protocol.
Pspax2
Manufactured by Hanbio Biotechnology
Sourced in China
The PSPAX2 is a laboratory equipment designed for the purification and isolation of plasmid DNA. It utilizes a silica-based membrane technology to efficiently extract and concentrate plasmid DNA from bacterial cell lysates. The core function of the PSPAX2 is to provide a reliable and high-yield method for the purification of plasmid DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2g, by Hanbio Biotechnology (9 mentions)
Lipofiter, by Hanbio Biotechnology (4 mentions)
Phb u6 mcs zsgreen puro empty, by Hanbio Biotechnology (2 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Cellulose acetate filter, by Merck Group (1 mentions)
Plasmid dna purification kit, by Macherey-Nagel (1 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Pcdh cmv mcs ef1 gfppuro, by System Biosciences (1 mentions)
9 protocols using pspax2
1
Generating ABCC5 Knockdown and Overexpression Cells
2
Lentiviral Transduction of miR-137 and SPHK2
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The 2nd generation system was used for lentivirus transduction. 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) were used as the interim cell line. Lentivirus packaging was performed by Hanbio Biotechnology Co., Ltd. in 293T cells using pSPAX2 (10 µg), pMD2G (5 µg), shuttle plasmids (10 µg) and Lipofiter (75 µl; cat. no. HB-LF-1000; Hanbio Biotechnology Co., Ltd.) for 16 h. A total of 48 and 72 h after transfection, lentiviruses were collected because more viruses are collected at two time points to ensure enough for the later experiments. Lentiviruses collected 48 and 72 h after transfection were both used in the subsequent experiments. Hblv-cmv-miR-137-GFP-puro, hblv-GFP-puro (control), hblv-cmv-SPHK2-GFP-puro [short hairpin RNA (sh)-SPHK2] and hblv-GFP-puro (NC) vectors (Hanbio Biotechnology Co., Ltd.) were used to transduce U251 and U373 cell lines at a multiplicity of infection of 3 at 37˚C for 24 h. A total of 72 h after transduction, puromycin (3 µg/ml; Gibco; Thermo Fisher Scientific, Inc.) was added to the culture medium of transduced cells to create stable cell lines for 7 days. The overexpression of miR-137 and knockdown of SPHK2 following transduction with lentiviral vector was detected according to the manufacturer's protocol.
3
Lentiviral-mediated Nrf2 Knockdown in HBE Cells
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Targeted oligonucleotides (5′-AGTTTGGGAGGAGCTATTATC-3′) were designed and cloned in pHBLV-U6-MCS-EF1-t2a-puro lentivirus RNAi vector (Hanbio, Shanghai, China). Then 293T cells were transfected with plasmid PSPAX2, PMD2G, and LipoFiter (Hanbio, Shanghai, China) to synthesize recombinant lentivirus. Supernatants containing lentivirus were collected 48 h after transfection, and filtered through 0.22-μm cellulose acetate filters (Millipore, USA). Then, the recombinant lentivirus was concentrated by super-centrifugation at a speed of 50,000g for 2 h.
In the knockdown experiment, HBE cells (0.5 × 106 cells/well) were cultured on 6-well plates. When HBE cells reached 60% confluence, the cells were treated with lentivirus (MOI 50) combined with polybrene (5 μg/ml) for 24 h, then replace the medium containing virus with fresh medium to the HBE cells. Most HBE cells (80%) were found to express EGFP 48 h after transfection. The negative control was the empty lentivirus vector lenti-EGFP. HBE cells were collected 3 days after virus infection and the Western blot analysis was performed to examine the expression of Nrf2.
In the knockdown experiment, HBE cells (0.5 × 106 cells/well) were cultured on 6-well plates. When HBE cells reached 60% confluence, the cells were treated with lentivirus (MOI 50) combined with polybrene (5 μg/ml) for 24 h, then replace the medium containing virus with fresh medium to the HBE cells. Most HBE cells (80%) were found to express EGFP 48 h after transfection. The negative control was the empty lentivirus vector lenti-EGFP. HBE cells were collected 3 days after virus infection and the Western blot analysis was performed to examine the expression of Nrf2.
4
CRISPR/Cas9-Mediated Knockout of CBS in Colon Cancer Cells
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The CRISPR/Cas9 system was used to achieve stable knockout of CBS in Caco-2 and HT-29 cells. In short, the interference target sgRNA was designed based on the sequence of the CBS gene as shown below: CTGATGAGATCCTGCAGCAG. We phosphorylated, annealed, and cloned the guide oligonucleotide into the BsmBI site of the pHBLV-U6-gRNA-EF1-CAS9-PURO vector (Hanbio Biotechnology, China) followed by verifying the constructed vector by sequencing. Then we transformed the transfer plasmid with guide oligonucleotide into Escherichia coli DH5α, and used Plasmid DNA purification kit (Macherey-Nagel, Germany) to isolate the plasmid from bacteria. Transferable lenti-CAS-puro plasmid (Hanbio Biotechnology, China), packaging plasmids psPAX2 (Hanbio Biotechnology, China) and pMD2G (Hanbio Biotechnology Co., Ltd., China) were transfected into 293 T cells to produce the lentivirus. 48 and 72 h after transfection, we collected the virus-containing supernatant, then transfected Caco-2 or HT-29 cells with the supernatant. Fresh medium containing puromycin (10 mg/L) was utilized for replacing the medium containing lentivirus after infected for 16 h. After 7 days of screening, we collected the puromycin-resistant cells. In the end, western blot was used to detected the cell infection efficiency.
5
Establishing Ovine Adipose Stem Cell Knockdown Model
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The encoding sequence of MEOX2 was ligated into the pHBLV-CMIVE ZsGreen-T2A-puro vector (Hanbio, Shanghai, China). We designed and synthesized two pairs of oligonucleotide sequences with short hairpin structures (shMEOX2-1 and shMEOX2-2), targeting the coding region of the MEOX2 gene. These oligonucleotides were then heat-treated to produce complementary double strands that were attached to the pHBLV-U6-ZsGreen-T2A-puro plasmid (Hanbio, Shanghai, China) between the BamHI and EcoRI restriction sites. The shRNA sequences for MEOX2 are shown in Table 4 . The recombinant plasmid and two packaged plasmids, PMD2.g and psPAX2 (both purchased from Hanbio, Shanghai, China), were transferred to 293 T cells. Forty-eight hours after transfection, the supernatant was collected and filtered through a 0.45-μm filter membrane. The filtrate was collected in a centrifuge tube. When the ovine SVFs density reached approximately 60%, recombinant lentivirus was used to transfect ovine SVFs. The medium was changed 24 h after infection. The cells in each group were collected 12 days after differentiation and subjected to qRT-PCR, western blotting, and ORO staining.
6
Investigating KIF20A Regulation in Ovarian Cancer
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Human ovarian surface epithelial (HOSE) cells and epithelial ovarian cancer cell lines (SKOV3, A2780, OV90, and OVCAR3) were cultured following the methods introduced previously [29 (link)]. Lentiviral KIF20A shRNAs (shKIF20A) were constructed by HanBio Technology, based on pLKO.1-puro plasmid. The following sequences were used: shKIF20A#1, 5′-CGTACACCATTCAAGGTACTA-3′, shKIF20A#2, 5′-GCTAGATGAAACAAGTCAATT-3′, shKIF20A#3, 5′- GCCACTCACAAATTTACCTTT-3’. Lentiviruses expressing myc-tagged BTRC (myc-BTRC), flag-tagged KIF20A (flag-KIF20A), and RELA (NF-κB p65) were generated based on the pLenti-puro backbone. pLenti puro HA-Ubiquitin (HA-Ub) (Plasmid #74218) was obtained from Addgene (Cambridge, MA, USA). Lentivirus for infection was produced by co-transfecting with the lentivirus packaging plasmids (pSPAX2 and pMD2.G, HanBio Technology) in 293T cells, as we previously described [30 (link)]. Cells were infected at a multiplicity of infection (MOI) of 15, with the presence of 6 μg/ml polybrene. Global protein translation inhibitor (cycloheximide, CHX) and the proteasome inhibitor MG132 were obtained from Selleck Chemicals. Ginsenoside Rg3 (purity>98%) was purchased from HerbSubstance (Chengdu, China).
7
Lentiviral Vector for PRRX1 Knockdown
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To generate PRRX1 low expression lentiviral vectors, we amplified the insert (full-length human PRRX1; NM_022716.2) by PCR from human reference cDNA. Lentiviruses were produced by transient transfection of 293 T cells with pSPAX2, pMD2G, and pHB-U6-MCS-zsgreen-puro (empty) (Hanbio, Shanghai, China) plasmid DNAs (2439 -BamHI and 2456 -EcoRI sites) plus LipoFiter™ (Hanbio Biotechnology, Shang Hai, China) following the manufacturer’s protocol.
8
Lentiviral Transduction of TRAF6 in 293T Cells
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The psuper‐retro‐puro shTRAF6 recombinant lentivirus (psuper‐TR1 and psuper‐TR2) and the empty psuper‐retro‐puro shRNA lentivirus (psuper) were constructed by our laboratory according to our previous research.14 The recombinant plasmid or empty vector was transfected with packaging plasmids pIK (Invitrogen) into 293T cells. The opening reading frame (ORF) of the human TRAF6 gene was PCR amplified and inserted into the lentiviral expression vector pCDH‐CMV‐MCS‐EF1‐GFP‐Puro (System Biosciences). The recombinant plasmid pCDH‐3×Flag‐TRAF6 or pCDH‐3×Flag‐TRAF6 124mut and the empty pCDH, lentivirus plasmid pMD2.G, and psPAX2 (Hanbio Biotechnology) were co‐transfected in 293T cells at 90% confluence.14 Puromycin was used to select cells with a stable expression of psuper‐TR1, psuper‐TR2, psuper, pCDH‐3×Flag‐TRAF6, pCDH‐3×Flag‐TRAF6 124mut and pCDH.
9
Construction of Camelid Immune sdAb Library
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A pHEN2-based phagemid vector was used in the construction of camelid immune sdAb library. The third-generation lentiviral transfer vector pHBLV-puro, lentiviral packaging vector psPAX2 and envelope vector pMD2.G were purchased from Han bio, China. pHBLV-puro vector contains a CMV promoter upstream a multiple cloning site (MCS). We modified pHBLV-puro vector by inserting a signal peptide right after the CMV promoter, and a his6 tag, a (G4S)5 linker and a GPI attachment signal sequence, the C-terminal 34 amino acid residues of DAF, after the MCS. The modified lentiviral vector was designated pHBLV-puro-GPI. Recombinant sdAb or sdAb-Fc genes were synthesized by GenScript (Nanjing, China) and cloned into pHBLV-puro-GPI vector via Sph I and Nhe I sites.
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