CombitTA-FUS-DDIT3 mice were generated by cloning the human FUS-DDIT3 cDNA into the Combi-tTA vector

May 26, 2017

olymerase was used for PCR. The primers specific to pig APOBEC3F were 59-TGG TCA CAG AGC TGA AGC AG and 59-TTG TTT TGG AAG CAG CCT TT. The semi-nested primer set used to detect plasmid-expressed pig APOBEC3F was 59-CCA AGG AGC TGG TTG ATT TC, 59-CTG GAG CAA TAC AGC GAG AG and 59-TAG AAG GCA CAG TCG AGG, with the latter being vector specific. The mammalian beta-actin primers were 59-CCT TCA ATT CCA TCA TGA AGT G and 59-CCA CAT CTG CTG GAA GGT. These primers amplify equally well a 236 bp beta-actin fragment from all mammals tested, including pigs and humans. Fluorescent microscopy The human APOBEC3G-GFP, pig APOBEC3F-GFP and GFP expression 14500812 constructs were described previously. The pig and human cell lines were maintained as above. One day prior to transfection, 5,00020,000 cells were seeded onto LabTek chambered coverglasses. After 24 hrs incubation, these cells were transfected with 250 ng of the relevant plasmid construct. After 24 hrs of additional incubation, images of the live cells were acquired using a Zeiss Axiovert 200 microscope at 4006 total magnification. Images were analyzed using Image J software. 18645012 Reverse transcriptase activity assays Whole cell protein extracts were DCC-2036 price prepared from 293T cells by suspending 500,000 cells in PBS, sonicating twice for 5 seconds and clarifying the lysates by centrifugation. Soluble protein levels were quantified using a BioRad Bradford assay. 10 mg of cell lysate was tested for reverse transcriptase activity using a C-type-RT activity assay following the manufacturers’ instructions. Cell-free PK-15 supernatants were assayed directly using the Cavidi Tech ELISA assay. PERV pol gene DNA sequence analyses Human 293T cell genomic DNA was prepared from terminal cocultures and 50 ng was used for high fidelity, PERV pol genespecific PCR reactions. 193 bp products were cloned using the Zero Blunt TOPO PCR Cloning kit and sequenced. Sequence comparisons were performed using Sequencher software and publicly available Clustal W alignment algorithms. APOBEC3G Blocks PERV Zoonoses expression of plasmid-derived pig APOBEC3F in PK-15 cells after 26 days of continuous co-culture. Non-transfected cells and diluted APOBEC3F plasmid DNA provided negative and positive controls, respectively. The larger 319 bp and smaller 190 bp bands are the specific PCR products of the first and second rounds of semi-nested PCR, respectively. A histogram summarizing the level of PERV transmission that was observed after 23 days of co-culturing human 293T cells with PK-15 cells expressing a vector control or over-expressing pig APOBEC3F. Two datasets, each with an independent PK-15 clone in three replica co-culture wells, were collected in parallel and averaged for each histogram bar. One standard error of the mean is shown. The experimental parameters are identical to those used in the 193 bp product shown in ACKNOWLEDGMENTS We thank Y. Blanchard, W. Brown, P. Hackett, E. Hendrickson, M. Hertzberg, L. Mansky, W. Mothes, M. Murtaugh and H. Schuurman for valuable feedback, M. Titus for microscopy facilities, J. Lingappa for antisera, G. Hache for assistance with cell culture, N. Somia for beta-actin primer sequences and M. Malim, A. Bielinsky and the ATCC for cell lines. Genetic Variation in Zoonosed PERV pol Gene Sequences. Sequences of the PERV pol gene fragments cloned from 293T cells co-cultured with control vector-expressing PK-15 cells. The number of times that each sequence was recovered is shown. Experiments 1 and 2 used geno