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Gv280 vector

Manufactured by Genechem

The GV280 vector is a molecular biology tool used for plasmid DNA manipulation and gene expression. It provides a standard platform for cloning and propagating genetic constructs. The core function of the GV280 vector is to enable the stable maintenance and replication of inserted DNA sequences in host cells.

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3 protocols using gv280 vector

1

Lentiviral Knockdown and Overexpression of miR-744

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Reverse complement sequence of miR744 mature was synthesized and subcloned into the AgeI/EcoRI site of GV280 vector (GeneChem, Shanghai, China) to generate the construct that inhibits miR744 expression, and this construct was named LVanti-miR744. The GFP vector was used for control. PC3 cells were transfected with LVanti-miR744, or control vector. Conversely, LNCaP cells stably overexpressing miR-744 vectors (GV209) or control vector constructed by GeneChem (Shanghai, China) were established by infection with lentivirus named LV-miR-744. Lentiviruses carrying overexpressing human NKD1 lentiviral vectors (GV358) were from GeneChem. The viruses were used to infect cells in the presence of Polybrene. After 48 hours, cells were cultured in medium containing puromycin for the selection of stable clones. The efficiency of knockdown and overexpression were determined by realtime quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analysis. The GFP vector was used for control.
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2

Overexpression of miR-455-5p in DU145 cells

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A complement sequence of mature miR-455-5p was synthesized and subcloned into the AgeI/EcoRI site of a GV280 vector (GeneChem, Shanghai, China) to generate a construct that overexpresses miR-455-5p. This construct was named LV-miR-455-5p. The empty vector (LV-miR-NC) was used as negative control. DU145 cells were transfected with LV-miR-455-5p or the control vector.
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3

Lentiviral Transduction of miR-874-3p and HDAC1

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miR-874-3p mimic non-targeting negative control (NC; 5'-ACUACUGAGUGACAGUAGA-3') or miR-874-3p mimic (5'-CUGCCCUGGCCCGAGGGACCGA-3') were inserted into the GV309 vector (Shanghai GeneChem Co., Ltd.); miR-874-3p inhibitor non-targeting NC (5'-CAGUACUUUUGUGUAGUACAA-3') or miR-874-3p inhibitor (5'-UCGGUCCCUCGGGCCAGGGCAG-3') were inserted into the GV280 vector (Shanghai GeneChem Co., Ltd.). For HDAC1 overexpression, the coding sequence of HDAC1 was inserted into the GV358 vector (Shanghai GeneChem Co., Ltd.). The empty vector was used as the NC for HDAC1 overexpression. 293T cells (American Type Culture Collection) were transfected with 20 µg the GV vector (GV309, GV280 or GV358) together with 15 µg pHelper 1.0 and 10 µg pHelper 2.0 vectors (both from Shanghai GeneChem Co., Ltd.) and then cultured at 37˚C in a humidified incubator in an atmosphere of 5% CO2 for 48 h to produce lentiviral particles. Transduction was performed when cells were growing exponentially and were 70-80% confluent. Polybrene (8 µg/ml, Sigma-Aldrich; Merck KGaA) and appropriate lentiviral particles (108 TU/ml, 5 µl) were added to cells for 16 h at 37˚C in a humidified incubator in an atmosphere of 5% CO2. The medium containing lentiviral particles were removed from wells and fresh medium were added. Subsequent experiments were performed 24 h later.
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