shRNA silencing was performed using lentivirus transduction for XBP1 or IRE1 (Sigma Mission shRNA lentiviral vector). Populations of transduced hSAECs were selected in 2 μg/ml puromycin. The most effective silencing lentivirus was identified by Q-RT-PCR and used in subsequent experiments. shRNA target sequences were: XBP1, 5’-GCCTGTCTGTACTTCATTCAA-3’; IRE1, 5’-GCAGGACATCTG GTATGTTAT-3’. Non-targeting luciferase shRNA lentiviral vector was used as negative control (Sigma, cat. SHC007).
Shrna lentiviral vector
Manufactured by Merck Group
The ShRNA lentiviral vector is a laboratory tool used to introduce short hairpin RNA (shRNA) into target cells. The shRNA is designed to knockdown the expression of a specific gene, enabling the study of its biological function. The vector provides a reliable and efficient method for gene silencing experiments in cell culture and animal models.
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7 protocols using shrna lentiviral vector
1
Silencing XBP1 and IRE1 in hSAECs
2
DNMT1 Silencing for Tumor Sphere Assay
3
Silencing PROX1 in PDLSCs Using Lentiviral Transduction
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An shRNA lentiviral vector targeting the CDS region of PROX1 mRNA (Clone ID: NM_002763.3-531s21c1) was purchased from Sigma-Aldrich. Lentiviral particles were prepared by transfecting HEK293T cells with pLKO.1-shPROX1 (or pLKO.1-shControl), psPAX2, pMD2.G plasmids at the ratio of 5:3:2. Media containing lentiviruses were harvested at 48 h and 72 h and used to transduce PDLSCs. Transduced PDLSCs were selected under 2 μg/mL puromycin for a week.
4
Knocking Down APE1 mRNA in BCBL-1 and PDLSCs
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An shRNA lentiviral vector targeting the 3’-UTR site of APE1 mRNA (Clone ID: NM_080649.1-1305s1c1) was purchased from Sigma-Aldrich. Lentiviral particles were prepared by transfection HEK293T cells with pLKO.1-shAPE17958 (or pLKO.1-shControl), psPAX2, pMD2.G plasmids in the ratio of 4:3:1. Media containing lentiviruses were harvested at 48 h and 72 h and used to transduce BCBL-1 cells and PDLSCs. Transduced BCBL-1 cells were selected under 2 μg/ml puromycin for a week, while KSHV-PDLSCs was transduced transiently and cells were used 72 h after transduction.
5
Lentiviral Knockdown and Overexpression of Genes in Renal Cells
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We generated the HK-2 stable Lsr knockdown cell line using shRNA lentiviral vector and puromycin (Sigma-Aldrich) selection. The shRNA hairpin oligonucleotides (TTTGAAGGAACACTGATGA), complementary to all the six variants of human Lsr mRNA sequences (NM_015925.7, NM_205834.4, NM_205835.4, NM_001260489.2, NM_001260490.2, and NM_001385215.1), were synthesized by Sangon Biotechnology Inc., and annealed and cloned into the lentivirus vector (pLKO.1) to create the Lsr shRNA construct. For the overexpression of CHRDL1, full-length sequence corresponding to the Chrdl1 transcript variant 1 (NM_001114385.1) coding region was cloned into the pLVX vector. For the overexpression of Lsr transcript variant 2 (NM_001164184), the coding region was cloned into the pLVX-IRES-ZsGreen1 vector. The lentiviral system was used to knock down Bmpr1a and Elavl1 and to overexpress CHRDL1 and LSR in the renal proximal tubular epithelial cells. The vector containing target sequence or the control vector was transfected into HEK293 cells along with the packaging plasmid lenti-VSV.G, pRSV-Rev, and pMDLg/pRRE. The protocol for concentrating virus is as described before. 17
The viral pellet was then resuspended in sterile PBS and used to infect cells at a fixed titer of 1×10 6 CFU/ml.
The viral pellet was then resuspended in sterile PBS and used to infect cells at a fixed titer of 1×10 6 CFU/ml.
6
Polycistronic Construct pMXs-puro-MGT
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The polycistronic construct pMXs-puro-MGT was constructed as previously described [14 (link)]. The plasmid map of pMXs-puro-MGT is provided in Figure
S1 . shRNA lentiviral vectors with pLKO.1 backbone were obtained from Sigma-Aldrich. Packaging and envelop vectors for lentivirus were psPAX2 and pMD2.G (Addgene).
7
Pvrl1 Knockdown in Mvt1 and 6DT1 Breast Cancer Cells
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shRNA lentiviral vectors were purchased from Sigma-Aldrich (cat. # SHCLNG-NM_021424). Stable Pvrl1 knockdown cell lines were generated by lentiviral transduction into Mvt1 cells and knockdown validated by qRT-PCR. 8x105 Mvt1 or 105 6DT1 cells were inoculated into the four mammary fat pad of 6–8 week old FVB/NJ female mice, 10 animals per group. Animals were euthanized at 5 weeks (experiment 1) or 4 weeks (experiment 2) after implantation. One-way Anova with Dunnett’s correction for multiple testing was performed for each experiment using GraphPad Prism. The results of the replicate experiments were then combined using Fisher’s combined probability test. All procedures were performed as approved by the NCI-Bethesda Animal Care and Use Committee.
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