Endofree plasmid maxi

Plasmids encoding active and mutant forms of a deactivated-Cas9-histone acetyltransferase P300 fusion protein (pcDNA-dCas9-p300 Core, pcDNA-dCas9-p300 Core-D1399Y) and guide RNA only expression vector (phU6-gRNA) were gifts from Charles Gersbach (Hilton et al., 2015 (link)) (Addgene #61357, #61358, and #53188, respectively). DER-specific gRNAs were designed by submitting DER coordinates or sequence to the CRISPOR¹ and Breaking-Cas (Oliveros et al., 2016 (link)) servers, respectively, and selected based on overlap with BEC DNAse hypersensitivity sites (ENCODE). Control gRNAs to IL1RN were described previously (Perez-Pinera et al., 2013 (link)). gRNAs were annealed and cloned into phU6-gRNA via BbsI restriction sites (NEB #R0539S). All plasmids were transformed into OneShot^®TOP10 competent cells (Thermo Fisher Scientific), cultured overnight, and extracted using Endo Free^® Plasmid Maxi or QIAprep^® Spin Miniprep (gRNAs) kits (QIAGEN). gRNAs sequence and locations are listed in Supplementary Table S5.

Intrabody expression plasmids were labeled with a C-terminal hemagglutinin (HA) epitope tag with amino acid sequence YPYDVPDYA to identify intrabodies. To direct the intrabodies and their cargo to the proteasome, a standard or scrambled PEST motif corresponding to amino acids 422–461 from mouse ODC (GenBank accession number NM_013614.2) was after the HA-tag. NbSyn2, NbSyn87 [19 (link)], and VH14 single domain (GenBank Accession number JX430807.1) nanobodies were subcloned with standard cloning techniques into pcDNA3.1- and pAAV-MCS according to the following cloning strategy: XbaI-intrabody-NotI-HA, XbaI-intrabody-NotI-HA-PEST, or XbaI-intrabody-NotI-HA-PEST-Scramble. α-Syn-(Gly₄Ser)₄-GFP, α-Syn-(A53T)-(Gly₄Ser)₄-GFP, α-Syn, and α-Syn(A53T) were subcloned into pcDNA3.1 at Nhe1 and HindIII restriction sites. Vectors for the expression of human mutant httex1-72Q as previously described were labeled with either GFP (pcDNA3.1-mhttex1-72Q-GFP) [20 (link)] or RFP (pcDNA3.1-mhttex1-72Q-RFP) [21 (link)]. All expression plasmids were verified by Sanger DNA sequencing and prepared with EndoFree Plasmid Maxi (Qiagen) prep kits according to manufacturer protocol.

cDNAs encoding the intrabodies in pAAV—C4 scFv (GenBank accession number EU490426) and V_L12.3—were used as previously described (Colby et al., 2004a (link),b (link); Kvam et al., 2009 (link)). For the nuclear export signal (NES) control experiments, DNA was polymerase chain reaction (PCR) amplified using complementary primers that encoded mitogen-activated protein kinase kinase NES. The resulting PCR product was ligated into pAAV-MCS (Stratagene) at the corresponding Xba1 and HindIII restriction sites using standard cloning techniques. The resulting expression plasmid cassette was the following: pAAV-MCS-Kozak sequence-C4 scFv-NES-HA-stop or pAAV-MCS-Kozak sequence-V_L12.3NES-HA-stop. Human Httex1 with 72 polyglutamine repeat lengths was labeled with enhanced green fluorescent protein, Httex1-72Q-EGFP, and expressed with pcDNA3.1(-) plasmid vector. To label nuclei in live cells, a NLS-tagged monomeric red fluorescent protein (RFP-NLS) described by Kvam et al. (2009) (link) was cotransfected in with Httex1-72Q-EGFP and intrabody. All expression plasmids were prepared using EndoFree Plasmid Maxi (Qiagen) and confirmed by DNA sequencing.

To generate plasmids for DNA vaccination, a previously described plasmid expression vector designated as pT.neo was used [26] (link). The antigenic peptide sequences were inserted at the 3′ EcoRI/XbaI flanking site of the T helper epitope of pT.neo. For co-immunization studies, a previously described plasmid encoding for murine interferon-γ (pmIFN-γ) was used [27] (link). The plasmids were produced by transforming DH5a Escherichia coli cells and purified, after sequence analysis, using EndoFree Plasmid Maxi or Giga kits (Qiagen). Each lot of plasmid DNA had a A₂₆₀/A₂₈₀ ratio ≥1.8, as determined by UV spectrophotometry, endotoxin content ≤0.1 EU/µg DNA, as determined by Limulus Amebocyte Lysate test (Associates of Cape Cod) and a predominantly supercoiled form.
For comparison, two selected peptides were conjugated to keyhole limpet haemocyanin (KLH) to increase their antigenicity and immunogenicity.

Cloning of genomic–cDNA hybrids of HGF and construction of expression vector pCK-HGF-X7 plasmid have been described in detail by Pyun et al. (12 (link)). All expression vectors used in experiments were purified by using an EndoFree plasmid Maxi or Giga Prep Kit (Qiagen, Hilden, Germany) dissolved in 0.9% NaCl, diluted to 2 mg/ml, and stored at −80°C before use.