Kod hot start polymerase

Detection of recombination with ERVs utilized primers recognizing M-MLV, polytropic, xenotropic ERV and the RT_universal_primer (Table in S3 Table) [92 (link)]. PCR analysis on genomic DNA of 293mCAT virus infected cells was performed using a combination of RT universal primer (5’ CCTACTCCGAAGACCCCTCGA-3’) and primers specific for polytropic and xenotropic ERVs (Table in S2 Table) using KOD Hotstart polymerase (Millipore, 71086) according to suggested parameters. PCR products from the reaction with RT_Universal_primer and Polytropic_JS5_rev on 293mCAT infected cells from TP^-6, 7 and 9 mice were cloned into pCR4-TOPO vector using the TOPO TA kit following the protocol provided by the manufacturer (Invitrogen, K4575-40). Recombinant plasmids were sequenced using the T3/T7 sequencing primers from the manufacturer, and 4981_fwd and MLV_IN_T159A_fwd to determine the 5’ recombination junction. PCR with Polytropic_JS5_fwd and 7791_reverse primers determined the 3’ recombination junction for the same 293mCAT samples. PCR analysis of genomic DNA from mice tumor or thymus samples required a nested PCR. First round PCR used the RT_universal_fwd primer and the MLV_LTR_U3_rev primer. Second round of PCR used a primer pair of 7791_reverse primer and the Polytropic_JS5_ fwd primer.

GFP-FLAG-USP42 and USP42 C120A have been described before (18 (link)). All deletion constructs were derived by PCR-based deletion using KOD Hot Start polymerase (Merck Millipore, 71842).

SOX9 expression vector was purchased from the plasmID Repository at Harvard Medical School (clone HsCD00004049). SOX9 cDNA was amplified from this vector and inserted into a pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA) using HindIII and XhoI restriction enzymes (New England Biolabs, Ipswich, MA, USA). DNA from melanoma cells for cloning of the CEACAM1 promoter was purified using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). Promoter fragments containing the full or partial putative promoter of CEACAM1 were amplified and cloned into pGL4.14 reporter vector (Promega, Madison, WI, USA) using XhoI and HindIII sites. Point mutations and deletions were introduced into the various constructs using specific primers, DNA synthesis with KOD Hot Start Polymerase (Merck Millipore, Darmstadt, Germany) and ultimately DpnI (New England Biolabs, Ipswich, MA, USA) digestion at 37°C for 1 hour. The full sequences of all primers used are detailed in Supplementary Table 1.

Primers HiaFULL-F and HiaFULL-R (see Table S1 in the supplemental material) were used to amplify full-length wild-type hia (R2866_0725) including the signal sequence (residues 1 to 49) from genomic DNA prepared from NTHi strain R2866. PCR was carried out using KOD hot-start polymerase (EMD Millipore) according to manufacturer’s instructions. Following digestion with BspHI and XhoI (NEB) and clean up, DNA was cloned into pET15b digested with NcoI and XhoI. The resulting plasmid was designated pET15b::Hia. Following confirmation of correct clones by sequencing, overexpression was carried out in E. coli BL21 following by inducing cells with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) overnight at 37°C with 200 rpm shaking. Overexpression was confirmed by Western blotting as previously described (23 (link)) using anti-Hia monoclonal antibody 1F4 (51 (link)). Whole-cell ELISA using standard methods (52 ) with modifications as previously described (23 (link)) and starting with 1:10,000 dilution of primary antibody anti-Hia monoclonal antibody 1F4 confirmed the location of Hia at the cell surface.

Inverse PCR was carried out using primer pairs designed to introduce point mutations as previously described and used here to abrogate binding of E. coli expressing Hia to Chang cells (22 (link)). D618K, A620A, and a 618/620 double mutant were generated using specific forward primers Hia-D618K-F, Hia-A620R-F, or Hia-618/620-double-F and common reverse primer Hia-618/620-R. A R674A mutant was generated using primer pair Hia-R674A-F and Hia-R674A-R. All inverse PCRs were carried out using KOD hot-start polymerase (EMD Millipore) according to manufacturer’s instructions, and a plasmid miniprep (Qiagen) of pET15b::Hia as the template. All primer sequences are listed in Table S1. Clones were sequenced using primers either side of the point mutation Hia-screen-F and Hia-screen-R using BigDye 3.1 according to manufacturer’s instructions (Thermo Fisher), and sequenced at Australian Genome Analysis Facility (AGRF, Brisbane, Australia). Overexpression was carried out as described above for the Hia wild type, and cell surface localization was confirmed using whole-cell ELISA as above.

The plasmid pKT3M (kindly provided by Dr. Upinder Singh, Stanford University, Stanford, CA, USA) [26 (link)] was used for construction of an ACK RNAi cell line. The full-length Entamoeba histolytica ACK coding sequence (EHI_170010) was PCR-amplified from E. histolytica genomic DNA using KOD Hot Start Polymerase (EMD Millipore, Billerica, MA, USA). The PCR product was cloned into AvrII and XhoI restriction sites to replace the resident luciferase (LUC) control gene. The final construct was confirmed by sequencing.
E. histolytica trophozoites were transfected with the ACK RNAi construct or the control pKT3M plasmid by electroporation as described previously [40 (link),41 (link)]. A total of 2.4 × 10⁶ cells were electroporated with 100 μg of DNA using two consecutive pulses at 1.2 kV and 25 uF and inoculated into TYI-S-33 medium. Transfectants were selected after two days by adding G418 to the medium to a final concentration of 6 μg/mL. Stable transfectants were maintained under this level of G418 selection. Primers used for construction of the ACK RNAi plasmid are listed Table 2.