Psuper retro system

Manufactured by Oligoengine

The PSuper-retro system is a specialized laboratory equipment designed for high-performance DNA and RNA amplification. It features a compact and user-friendly interface, enabling precise control over thermal cycling parameters for optimized nucleic acid amplification.

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PSuper-Retro (5 citations) PSUPER system (3 citations)

2 protocols using psuper retro system

Plasmid construction for Plac8 KD and overexpression

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Plasmids for retro-viral Plac8 KD, and shRNA-resistant Plac8 expression were described previously (McMurray et al., 2008 (link)). cDNAs for Rab7, Rab5a, and Atg12 were PCR-cloned in pBabe-hygro containing an in-frame, N-terminal 3xFlag-tag and sequence verified. Dominant-negative and activated Rab7 and Rab5a mutants were generated via site-directed mutagenesis and sequence-verified. N-terminal 3xFlag tagged DN Rab7, DA Rab7, DN Rab5a and Atg12 were PCR-cloned in pLenti/Ubc/V5 (Invitrogen) and sequence-verified. cDNA for LC3 was PCR-cloned in pEGFP (Clonetech) and subsequently PCR-cloned into lenti-viral vector FG12. The mCherry-EGFP-LC3 puro construct (Addgene 22418) (N’Diaye et al., 2009 (link)) was cloned into pBabe hygro. Atg12 shRNA molecules were designed (http://jura.wi.mit.edu/bioc/siRNAext/home.php) and cloned into the pSuper-retro system (Oligoengine). Atg12 lenti-viral KD constructs were obtained from Openbiosystems.

Kinsey C., Balakrishnan V., O’Dell M.R., Huang J.L., Newman L., Whitney-Miller C.L., Hezel A.F, & Land H. (2014). Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression. Cell reports, 7(4), 1143-1155.

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Immortalized IMR90 Lung Fibroblast Knockdowns

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IMR90 primary human lung fibroblasts (ATCC), previously hTERT immortalized (20 (link)), were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Cell lines depleted of macroH2A1 or luciferase as a control were generated by retrovirus-mediated expression of shRNA using the pSuper.Retro system (OligoEngine). The targeting sequence for macroH2A1 was 5′- GCGTGTGTTGTGGTGCTTTAT-3′. The targeting sequence for macroH2A1.1 was 5′- GGCGACAAACACTGACTTCTA-3′. The targeting sequence for macroH2A1.2 was 5′- CTGAACCTTATTCACAGTGAA-3′. The luciferase shRNA targeting sequence was 5′-GATATGGGCTGAATACAAA-3′. The cells were selected and maintained under 0.5 mg/ml G418 in MEM supplemented with 10% FBS.

Ruiz P.D., Hamilton G.A., Park J.W, & Gamble M.J. (2019). MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair. Molecular and Cellular Biology, 40(1), e00230-19.

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