A 21-nucleotide shRNA construct targeting mouse SRA1 mRNA was cloned into the retroviral pSUPERIOR.retro.puro vector (OligoEngine (Seattle, WA)) or the pLentiLox3.7-GFP vector with a sense-loop-antisense design, using the loop sequence CTTCCTGTCA as described [29] (link).
Psuperior retro puro vector
Manufactured by Oligoengine
PSUPERIOR.retro.puro vector is a laboratory equipment used for vector cloning and expression. It provides a platform for the insertion and propagation of genetic sequences of interest. The core function of this product is to facilitate the molecular biological processes involved in recombinant DNA technology.
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3 protocols using psuperior retro puro vector
1
Targeting mouse SRA1 mRNA via shRNA
2
FoxO3a siRNA Knockdown in KGN Cells
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The pSuperior.retro.puro vector (OligoEngine) was used for the expression of siRNA in KGN cells. FoxO3a siRNA vector was generated by a gene-specific insert (5′-GTGGAGCTGGACCCGGAGT-3′) to target FoxO3a. A negative control (NC) vector was constructed using an insert (5′-GTGTCTGTAGGAGTCATCC-3′) with no significant homology to any mammalian gene sequence. Transfection was performed using LipofectAMINE 2000, according to the manufacturer’s protocol. Briefly, KGN cells were transfected with 10 nM FoxO3a siRNA or NC siRNA for 48 hrs, followed by further analysis. FoxO3a levels in KGN cells were assessed by immunoblots after transient transfection. NC siRNA was used to assess non-specific gene-silencing effects.
3
Cloning and Expression of LysRS and cGAS
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shRNAs targeting LysRS and STING were cloned into the pSUPERIOR.retro.puro vector from Oligoengine, according to the manufacturer’s instructions. N-terminally Flag- and HA-tagged codon-optimized mouse LysRS (MWG Eurofins) was cloned into the pOZ retroviral vector. For bacterial expression of recombinant proteins, the human LysRS gene was amplified by PCR from complementary DNA (cDNA) of HeLa cells. The cDNA and codon-optimized human cGAS (MWG Eurofins) were cloned into the pGEX-4T1 plasmid. Codon-optimized mouse LysRS was cloned into the pGEX-4T1 plasmid. Danio rerio LysRS (zLysRS) was amplified by PCR from the cDNA of zebrafish larvae and cloned in pGEX-4T1.
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