Antiviral restriction factor transgenesis in the domestic cat
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1 Nature Methods Antiviral restriction factor transgenesis in the domestic cat Pimprapar Wongsrikeao, Dyana Saenz, Tommy Rinkoski, Takeshige Otoi & Eric Poeschla Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Lentiviral vectors Additional animal images and radiographs GFP and rhtrimcyp expression in PBMCs Additional films and images for whole body analysis. rhtrimcyp expression in whole organs Domestic cat embryo cleavage and blastocyst rates, % transgene expression, effect of timing Transgenic embryo development with single vs dual-gene vectors (all pre-ivf microinjections) Vector insertion sites
2 Supplementary Figure 1. Lentiviral vectors. TSinG and derivative TSinT2AG both utilize a standard 0.52 Kb internal hcmv promoter. For TSinT2AG, a porcine teschovirus 2A peptide (P2A) mediates expression of both proteins from a single precursor polypeptide. Proper co-translational cleavage at the 2A site was confirmed by immuoblotting. The other two vectors utilize a dual promoter having a minimal (0.16 Kb) hcmv core promoter (mcmv) and a PGK promoter. The four otherwise contain standard transfer vector elements: packaging signal, RRE, WPRE, cppt-cts (cppt); all are U3-deleted ( U3). See Methods for further details.
3 2a Supplementary Figure 2. Additional animal images and radiographs (panels b-d on subsequent pages). (a) 5.5 month images corresponding to Fig. 2A, with control and transgenic cat in the same frame. (b) Simultaneous control cat images for day 2. (c) Enlargements of the 30 day images in Fig. 2a. (d) Films were taken on day 45 of a pregnancy that terminated in spontaneous miscarriage 6 days later (term is days in this species).
4 2b
5 2c 2d
6 a Supplementary Figure 3. GFP and rhtrimcyp expression in PBMCs. of (a) Fluorescence microscopy PBMCs. Animall ages are shown at left. Small and large size bars represent 500 and 200 µm respectively. (b) rhtrimcyp; cells are also imaged for GFP and nuclear DNA (DAPI). Immunofluorescence for HA-tagged rhtrimcyp was done with rat anti-ha. Note that fewer cells expressed detectable TRIMCyp than GFP, and the two proteins were discordant to a large extent, suggesting the mcmv and PGK promoter elements in the vector are differentially active in subsets. b
7 b Supplementary Figure 4. Additional films and images for whole body analyses. (a) Uncropped versions (full films) of the immunblots shown in Fig 5a. (b) Integument (ventral aspect of chest and abdomen) of TgCat4 photographed under (i) ambient light and (ii) blue light illumination for GFP.
8 Supplementary Figure 5. rhtrimcyp expression in solid organs. Immunoblotting with anti-ha was performed as in Fig. 3A.
9 Supplementary Table 1. Domestic cat embryo cleavage and blastocyst rates, % transgene expression, effect of timing Treatment Number of oocytes Cleavage rate (%) Blastocyst rate (%) Embryo GFP expression (%) Mosaicism rate None ± ± Pre-IVF lentiviral vector injection ± ± ± 11.2 < 1% Post-IVF lentiviral vector injection ± ± ± % 1
10 Supplementary Table 2. Transgenic embryo development with single vs. dual-gene vectors (all pre-ivf microinjections) Lentiviral vector Proteins or proteins encoded in vector Promoter Number of oocytes Cleavage rate (%) Blastocyst rate (%) Embryo GFP expression (%) TSinG GFP hcmv ± ± ± 10.2 TBDmGpT GFP, rhtrimcyp mcmv!pgk ± ± ± 12.0 TBDmGpR GFP, RFP mcmv!pgk ± ± ± 9.5 TSinT2AG rhtrimcyp-p2a-gfp hcmv nd nd nd nd n.d.: not done. hcmv: human cytomegalovirus immediate early gene promoter. mcmv: minimal hcmv promoter. 2
11 Supplementary Table 3. Vector insertion sites Host - vector junction 1 chrun_acbe : 891-1, % Identity GGGTCTGTTCTTCCCATGATCTTCTTCCCCTGGGTACTTGATTCAGTGACTACTTTATAGGAGTTTAGCTG AGTTTCCTTCAGTGATTTAGGTTTATGGTGAATGTCTTTAACGTTTATTTATTTTTGGGGGGACAGAGAGA GACAGAGCATGAACGGGGGAGGGGCAGAGAGAGAGGGAGACACAGAATCGGAAACAGGCTCCAGGCTCAGA GCCATCAGCCCAGAGCCTGACGCGGGGCTCGAACTTGGAAGGGCTAATTCACTCC Host - vector junction 2 chrx: 127,955, ,956, % Identity GGGCATCATCAGCAGCACAGTCCTGTAAACGTTCTTGGATGTGGTTGGCACCCGGCCTTTGCTCGGTCAGA GTGGGTCAAAGCGGTTCCTCAGAAGTGAGGGGTCAGGAAACACAGTCACATCTGAGATAAAAGGAGGGAGG TGCTGCCTGGGCTTGGTCATGGACAGTGTAAAAGTGGGAATGGGTGGAAGCCAGAGACAAAGGATGGGTGC GTGATTGCTGGTGGGGGAGAACAGAGTTCCGATACTAGAGACTGGGTAGCTGGGTGACATCATTTTTAATC CTCCTGCGCGTGCGCGCAGGCACCCACAAGCACCACAACAATCCACCCCAGTAAGCTAAGAAGCACCTTCT AGTGGAGAACGGAGCCTGAGCCAACCTCACTTTTCATGAACACCACAAGTCTCTCTGCCTGCTTAGTTCAT GGACTATAAAGGGCTTCATAGTTTGACTTCTATGGGAAAATGATGTAATTTCAATTGTATTTCAGTTTGTT CGCTGGCCCATCTATTAAATTTCTTTATCTCTTTTTTGTTTCTTTTCTTTTTCTTGAATACAGAAGATAAA AAAAATATTTTTATTTTTAATTTTTATTAAAATATTTTGCTTTAATTTTTTCTACTTTATTTTTTACTTTT TTGTAAATTTTATAAATTCTAATTTAATTCCATCATTTCATTTTATCCTATTTCATTGTATTCATTTTTCA NATTTTCAAACATTTTACTTTTTTTATTCTTTTCTTACCTTTCCCTTTTAAATTTTTTTTAACATTTATTT ATTTTTGAGACAGAGAGAGACAGAGCATGAATGGGGGAGGGGCAGAGAGAGAAGGAGACACAGAATCAGAA ACAGTCTCCAGGCTCTGAGCTGTCAGCCCAGAGCCTGACGTGGGGCTCGAACTCATGGACCGCGAGATCGT GACCTGGCTGAAGTCGGACGCTTAACCGACTGCGCCACCCAGGCGCCCCTACCTTTCCCTTTTTAATCTAA TCTATAAAGCTCCTTTTGGAAGGGCTAATTCACTCC Host - vector junction 3 chrd1: 2,438,470-2,438, % Identity CCAGTAAGCTAAGCAGCACCATCTAGTGGAGAACGGAGCCATTACACTAAGCCCCCTCGATCTTGGCCTGG GCCAACCTCGATCTTCAGGAACACCACATGGAAGGGCTAATTCACTCC Host - vector junction 4 chrb1: % Identity CCAGTAAGCTAAGCAGCACCATCTAGTGGAGAACGGAGCCATTACACTAAGCCCCCTCGATCTTGGCCTGG GCCAACCTCGATCTTCAGGAACACCACATGGAAGGGCTAATTCACTCC Underlined sequences are the U3 terminus of the vector.
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