Stbl2 competent cells

Starting from our original pHAGE-Ubc-NLS-MCP-YFP third generation lentiviral vector (Lionnet et al., 2011a (link)), we swapped the YFP with the HaloTag open reading frame (DeJesus-Hernandez et al., 2011 (link)) to generate the pHAGE-Ubc-NLS-MCP-HaloTag. To achieve this, a HaloTag ORF fragment was generated by PCR and inserted into the digested pHAGE-Ubc-NLS-MCP-YFP vector by ligation. We used the pHAGE-Ubc-NLS-MCP-HaloTag plasmid to create recombinant lentiviral particles (Mostoslavsky et al., 2006 (link)) generating expression of NLS-MCP-HaloTag driven from the human ubiquitin C promoter in target cells.
The alpha-Amanitin-resistant RPB1 fused to Dendra2 (Dendra2-RPB1Amr) ORF was excised from our original Dendra2-RPB1Amr expressing vector using HpaI and NheI (Cisse et al., 2013 (link)). We digested the PB53x EF1 Series Piggybac vector (System Biosciences, Palo Alto, CA) with EcoRI, generated blunt ends and performed a second digestion with NheI. The ORF insert was then ligated into the vector using the TaKaRa DNA ligation kit LONG (TaKaRa Bio-Clontech, Shiga, Japan) following the blunt end ligation protocol. Constructs were transformed into Stbl2 competent cells (Life Technologies, Carlsbad, CA), resistant colonies were then screened by restriction analysis and confirmed by sequencing.

AAV9:Tnnt2-Cre and AAV:Tnnt2-Luc were constructed by placing codon-optimized Cre [14 (link)] or Luciferase into an ITR-containing AAV plasmid (Penn Vector Core P1967) harboring the chicken cardiac TNT promoter. AAV constructs were propagated in Stbl2 competent cells (Life Technologies; Cat. # 10268–019), and their integrity was validated by restriction digestion and DNA sequencing. AAV9 was packaged at the Viral Vector Core of the Gene Therapy Center, University of Massachusetts Medical School. Viral titer was determined by quantitative PCR. AAV:Tnnt2-Cre contained IRES-TdTomato, although the level of TdTomato expression was not detectable by microscopy. Aliquots were stored at -80°C prior to use.

In order to synthesize the miR-187-3p and SPRY1 inserts, reverse transcription (RT) was first performed with Superscript III reverse transcription reagent (Invitrogen), which was then used to amplify the miR-187-3p and SPRY1 inserts via polymerase chain reaction using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and the following primers: miR-187-3p EcoR1 forward primer (5′-CCGGAATTCCACCTGGAGCACAGGTCATC) and miR-187-3p-BamH1 reverse primer (5′-CGCGGATCCAGCTGTGTACGGAGAGACGA); or SPRY1-Not1 forward primer (5′-CAAAAAGCGGCCGCATGGATCCCCAAAATCAACATG) and SPRY1-Kpn1 reverse primer (5′-GGGAAAGGTACCTCATGATGGTTTACCCTGACC). The PCR product was purified, digested with restriction enzymes, and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP (Biosciences, Cambridge, UK). The plasmids were transformed into Stbl2-competent cells (Life Technologies, Carlsbad, CA, USA), and the sequence of positive clones was confirmed by DNA sequencing.