MVV vector integration in sheep cells

To test if LEDGF/p75 influences distribution of Maedi-visna virus (MVV) integration sites, we infected sheep CPT3, LKO1 (PSIP1-null), LHKO1 and LHKO2 (PSIP1/HDGFL2-null) cells with MVV-derived vector. Genomic DNA was isolated from infected cells, and chromosomal junctions at integrated U5 vDNA ends were amplified using linker-mediated PCR, sequenced using Illumina technology and mapped to sheep genome. Genomic DNA isolated from cells 5 days post-infection with MVV vector was processed for linker-mediated PCR, and the amplified viral LTR-chromosome junctions were sequenced. To this end, genomic DNA, digested with MseI overnight at 37°C, was ligated to a double-stranded DNA linker containing 5'-TA overhang overnight at 12°C. Customized DNA linkers were used in conjunction with barcoded primers to aid in multiplexing and prevent crosstalk between samples. The first round and the nested MVV U5 primers were 5'-CTAATTCCGTGCAACACCG and 5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC TNN NNN NCA ACA CCG GAG CGG ATC, respectively (including the 6-nucleotide barcode for multiplexing). The nested PCR primers contained Illumina adaptor sequences appended at the 5' ends. The PCR products, multiplexed on a single lane of a flow cell, were subjected to 150-bp paired-end sequencing on a HiSeq-4000 Illumina instrument.