Ubi mcs sv40 egfp ires puromycin

Manufactured by Genechem

Sourced in China

The Ubi-MCS-SV40-EGFP-IRES-puromycin is a lentiviral vector that contains a constitutive ubiquitin promoter, a multiple cloning site, an SV40 promoter, an EGFP reporter gene, an IRES element, and a puromycin resistance gene. This vector can be used for expression of transgenes in mammalian cells.

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4 protocols using ubi mcs sv40 egfp ires puromycin

Lentiviral Transduction for Gene Modulation

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Virus packaging was performed in HEK293T cells by co-transfection with lentiviral vectors with the packaging plasmid pHelper 1.0 vector (GeneChem Co., Ltd., Shanghai, China) and the envelope plasmid pHelper 2.0 vector (GeneChem Co., Ltd.) using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, supernatants containing lentiviral particles were collected, and the virus titer was quantified according to the manufacturer’s instructions. Lentiviral vectors encoding short hairpin RNAs (shRNAs) (Sh-1: ATTATTGTAACCACCCGTT; Sh-2 TGAGCAGTATTCCGAGAAA; Sh-3: CAATCATTTCATTGATCTT) targeting OGT were generated using the GV344 vector (hU6-MCS-Ubiquitin-Luc_firefly IRES-puromycin, GeneChem Co., Ltd., Shanghai, China). A scrambled GV344 vector (TTCTCCGAACGTGTCACGT) was used as the negative control. Stable transfectants overexpressing OGT were generated by lentiviral transduction using a GV341 vector (Ubi-MCS-3FLAG-SV40-puromycin, GeneChem Co., Ltd.). An empty vector was used as the negative control. Stable transfectants overexpressing EZH2 were generated by lentiviral transduction using a GV367 vector (Ubi-MCS-SV40-EGFP-IRES-puromycin, GeneChem Co., Ltd.). An empty vector was used as the negative control.

Jiang M., Xu B., Li X., Shang Y., Chu Y., Wang W., Chen D., Wu N., Hu S., Zhang S., Li M., Wu K., Yang X., Liang J., Nie Y, & Fan D. (2018). O-GlcNAcylation promotes colorectal cancer metastasis via the miR-101-O-GlcNAc/EZH2 regulatory feedback circuit. Oncogene, 38(3), 301-316.

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Modulating EBV-miR-BART8-3p Expression in Nasopharyngeal Carcinoma Cells

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Lentiviral particles (GV369 and Ubi-MCS-SV40-EGFP-IRES-puromycin) containing EBV-miR-BART8-3p precursors and lentiviral particles (GV280 and hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) containing reverse complement of EBV-miR-BART8-3p and their control vectors were constructed by Shanghai Genechem Co., Ltd. (Shanghai, China).CNE-1 and SUNE-1 cells were transfected with a recombinant lentiviral vector GV369 to upregulate EBV-miR-BART8-3p expression (CNE-1-BART8-3p and SUNE-1-BART8-3p cells), and C666–1 cells were transfected with a lentiviral vector GV280 to downregulate EBV-miR-BART8-3p expression (C666–1-BART8-3p cells). The transfection efficiency was checked using qPCR assay.
For the rescue assay, CNE-1-BART8-3p cells and SUNE-1-BART8-3p cells were transfected with the RNF38 lentiviral vector GV358 (Shanghai Genechem Co., Ltd.; Shanghai, China) or a normal control (Shanghai Genechem Co., Ltd.; Shanghai, China).

Lin C., Zong J., Lin W., Wang M., Xu Y., Zhou R., Lin S., Guo Q., Chen H., Ye Y., Zhang B, & Pan J. (2018). EBV-miR-BART8-3p induces epithelial-mesenchymal transition and promotes metastasis of nasopharyngeal carcinoma cells through activating NF-κB and Erk1/2 pathways. Journal of Experimental & Clinical Cancer Research : CR, 37, 283.

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Lentiviral Transduction for Tumor Cell Manipulation

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A lentivirus carrying miR-188-3p (UBI-MCS-SV40-EGFP-IRES-puromycin) and the red fluorescent protein gene was purchased from Gene Chem (Shanghai, China). Specifically, the plasmid carrying miR-188-3p was transfected into tumor cells and grown for 14 days to establish the puromycin-resistant cell population. Moreover, a lentivirus carrying CBL cDNA using the Ubi-MCS-3FLAG-CBh-CHERRY-IRES-puromycin vector and an Ubi-MCS-3FLAG-CBh-CHERRY-IRES-puromycin empty vector was also obtained from Gene Chem and used to establish a stably transfected cell population according to the manufacturer’s protocols. The stably transfected cell population was used for the in vivo assay in nude mice.

Lin J.J., Luo B.H., Su T., Yang Q., Zhang Q.F., Dai W.Y., Liu Y, & Xiang L. (2023). Antitumor activity of miR-188-3p in gastric cancer is achieved by targeting CBL expression and inactivating the AKT/mTOR signaling. World Journal of Gastrointestinal Oncology, 15(8), 1384-1399.

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Lentiviral Knockdown of lncRNA RP11-241J12.3

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Lentivirus (Ubi-MCS-SV40-EGFP-IRES-puromycin, Shanghai GeneChem, China) overexpressing RP11-241J12.3 (Lv-241J12.3) and control (Lv-control). Short hairpin RNAs (shRNAs) were specifically designed to knockdown lncRNA RP11-241J12.3. Two shRNAs targeting LncRNA241J12.3-4 and negative control shRNA were designed and cloned into plasmid vectors (pGPU6/GFP/Neo or pGPU6/Neo, GenePharma, Shanghai, China). These shRNAs were designated sh-241J12.3-4, sh-241J12.3-120, and sh-NC, respectively. The infection and transfection efficiencies were confirmed by qRT-PCR. Recombinant adenovirus (Ad-HBx) expressing HBx was prepared as described previously [13 (link)]. Empty Ad-N served as a control adenovirus. The shRNA sequences are shown in the supplementary data (Information S1).

Cheng L., Hu S., Ma J., Shu Y., Chen Y., Zhang B., Qi Z., Wang Y., Zhang Y., Zhang Y, & Cheng P. (2022). Long noncoding RNA RP11-241J12.3 targeting pyruvate carboxylase promotes hepatocellular carcinoma aggressiveness by disrupting pyruvate metabolism and the DNA mismatch repair system. Molecular Biomedicine, 3, 4.

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