Short hairpin RNA (shRNA) sequences targeting Dnmt1, Dnmt3a, or Dnmt3b mRNA were designed using the shRNA Sequence Designer (www.clontech.com ). Three shRNA sequences targeted against each of the DNA methyltransferase RNA sequences (Supplementary Table 3 ) were inserted into the Xho1 and HindIII sites of pSingle-tTS-shRNA (Clontech, Mountain View, CA). Presence of inserts was determined by restriction digestion with MluI (Promega, Madison, WI). Transfection, selection of stably transformed cells, and induction of shRNA expression with doxycycline (Dox; Clontech) was as described (35 (link)). A scrambled sequence inserted into pSingle (35 (link)) was used as a control.
Psingle tts shrna
Manufactured by Takara Bio
Sourced in United States
The pSingle-tTS-shRNA is a plasmid vector designed for the expression of short hairpin RNA (shRNA) in mammalian cells. It features a tetracycline-inducible promoter system for controlled gene silencing.
Automatically generated - may contain errors
4 protocols using psingle tts shrna
1
Knockdown of DNA methyltransferases
2
Inducible Silencing of RepID in Melanoma
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Specific silencing of endogenous RepID was achieved using an inducible shRNA-expressing vector, pSingle-tTS-shRNA (Clontech). shRNAs were inserted into the plasmid using the XhoI and HindIII cloning sites and were delivered into 2451 13T melanoma cells48 (link). Stable clones were selected, and cells conditionally expressing shRNA directed against RepID were induced (or not) with doxycycline for 16 days.
3
Claudin-5 Knockdown via AAV-2/9
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shRNAs designed to target transcripts derived from mouse claudin-5 were incorporated into adeno-associated virus (AAV)-2/9 vectors. shRNA was cloned into the pSingle-tTS-shRNA (Clontech, Mountain View, CA, USA) vector. The plasmid incorporating the inducible system with claudin-5 shRNA was digested with BsrBi and BsrGI, and ligated into the Not1 site of the plasmid pAAV-MCS, such as to incorporate left and right AAV-inverted terminal repeats. (L-ITR and R-ITR). AAV-2/9 was then generated using a triple transfection system in a stably transfected HEK-293 cell line for the generation of high-titre viruses (Vector BioLabs, Malvern, PA, USA).
4
ACTR5, Myocd, and Mef2c Overexpression and Knockdown
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The coding regions of human ACTR5, the cardiac and smooth muscle isoforms of mouse Myocd, and Mef2c were amplified by PCR and inserted into the pCAGGS and pCS2+ expression vectors. A tetracycline‐inducible shRNA vector, pSingle‐tTs‐shRNA (Clontech), was used to isolate cell lines exhibiting Actr5 knockdown under DOX control. The target sequence for the Actr5 knockdown was gacagatggaccagtttca, and the hairpin loop sequence was ttcaagaga. The AAV6 expression vector encoding Actr5 was constructed using the AAVpro Helper Free System (AAV6) (TAKARA BIO, Shiga, Japan). Purification and titration of AAV6 particles were performed using the AAVpro Purification Kit Maxi (All Serotypes) (TAKARA BIO) and AAVpro titration (TAKARA BIO) kits.
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