Nanojuice transfection kit

Plasmids expressing lamin A and lamin B1 fused to GFP were a kind gift from Tom Misteli [38 (link)] and pEGFP-KASH2 and pEGFP-KASH2ext were a kind gift from Didier Hodzic [39 (link)]. Gelsolin was cloned by RT-PCR: for reverse transcription, total RNA was isolated from B16–F10 by the NucleoZOL kit (740404.200, MACHEREY-NAGEL, Düren, Germany) according to the manufacturer’s instructions and reverse-transcribed using GoScript™ Reverse Transcription System (Promega) and the oligonucleotide 5′-GAGAACTGAAACCTGGGT-3′. The N-terminal part of Gelsolin (aa 1–370) was amplified by PCR using KOD Hot Start DNA Polymerase (71086, Merck, Kenilworth, NJ, USA) and the oligonucleotides 5′-cagctagccaccATGGTGGTGGAGCACCCC-3′ and 5′-gcaccggtaTGGGGTACTGCATCTTGGAGAT-3′. The PCR product was ligated into NheI-AgeI sites in pEGFP-KASH2ext upstream to GFP to generate pGSN-EGFP-KASH2ext. Transfection of cells was carried out by the Nanojuice transfection kit (71900-3, Merck, Kenilworth, NJ, USA) following the manufacturer’s instructions.

Knock-down of gene expression was achieved by introduction of siRNA duplexes (Sigma-Aldrich) designed for the draxin receptors, DCC and neogenin, into rat HNSPCs using the Lipofectamine RNAiMax Reagent (Invitrogen) according to the instructions. The sequences of siRNA duplexes used in this study were: 5′-CAGUGAACGGCUCCCAUAATT-3′ and 5′-UUAUGGGAGCCGUUCACUGTT-3′ for rat neogenin; and 5′-CUAUGUAUUACUUUCGAAUTT-3′ and 5′-AUUCGAAAGUAAUACAUAGTT-3′ for rat DCC. Overexpression was achieved by introduction of the expression plasmids described above into rat HNSPCs using the NanoJuice Transfection Kit (Merck-Millipore) according to the instruction.

Sequences from the two heptad peaks found in the Gpr56 locus (Wilson et al., 2010 (link)) were synthesized and cloned by the GenScript Gene Synthesis Service into a pGL4.10[luc2] reporter plasmid (Promega), with additional 50-bp sequences upstream and downstream to ensure proper peak coverage. The pGL4.74[hRluc/TK] Renilla reporter plasmid that was used to control for transfection efficiency was obtained from Promega. ESCs were plated on gelatin-coated plates (0.1% gelatin [BDH] in PBS for 20 min) and passaged twice to remove the feeders. Cells were then seeded in gelatin-coated 96-well plates at a density of 5,000 cells per well. Next day, the medium was changed and the cells were transfected with the Firefly luciferase reporter plasmids together with the Renilla luciferase reporter plasmid using the NanoJuice Transfection kit (Merck Millipore). After 24 hr, cells were treated with doxycycline (Sigma) for 24 hr and luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) and the POLARstar Omega device (BMG Labtech) following manufacturer's instructions.