E filtered out using custom Perl scripts with threshold Q20. All of the filtered sequencing reads were then mapped to the reference Bos taurus genome sequence (Bos_taurus_UMD_3.1, build 6.1), the hLF BAC (151,726 bp) and the pBeloBAC vector sequence (Genbank accession number: U51113, 7,378 bp), respectively, with Burrows-Wheeler Aligner (BWA, version 0.5.9) [18]. The unmapped reads were de novoFigure 1. Schematic representation of the transgene constructs. (A) Structure of the transgene construct released from the pBeloBAC vector by NotI digestion. The transgene backbone contains the lacZ gene, the chloramphenicol resistance gene (Cmr) and the regulatory genes repE, parA and parB. The dotted lines indicate the insertion position of the hLF BAC into the pBeloBAC vector. (B) The hatched, gray and open boxes represent the 59 regulatory, encoding and 39 regulatory regions, respectively, of the hLF gene. doi:10.1371/journal.pone.0050348.gReliable Method for Transgene IdentificationFigure 2. The transgene integration sites. The integration sites for the hLF BAC transgene on chromosome 15 in the bovine genome. (A) The left 58-49-1 supplier boundary of the integration site. (B) The right boundary of the integration site. Reference, the genomic DNA sequence of wide-type bovine; Transgene, the genomic DNA sequence of transgenic cattle; hLF, the DNA sequence of hLF BAC. The underlined regions indicate the deletion of 11 nucleotides in the genome. doi:10.1371/journal.pone.0050348.gassembled by SOAPdenovo (version 1.05) and the resultant contigs were blasted against the hLF transgene and pBeloBAC vector to find bridging reads between the host genome and the foreign fragments. To identify the interval of transgene integration, abnormal read pairs with one end mapping to the reference and the other end to the transgene or the vector 18325633 were selected for further security. The exact integration breakpoints were finally identified by split-read analysis that spanning the transgene insertion junctions.PCR AnalysisTo verify the integration breakpoints, PCR was performed on genomic DNA samples from the three transgenic cattle and a single wild-type cow. The 59 flanking transgene locus was identified using the primers G1 (59-CCCAGGCAACCATTAATCAG-39), G3 (59-ATGCCGTTGTTGACGTTGTA-39) and T2 (59-CTTAGCCCATGCC TCATTGT-39); the 39 transgene locus was identified using the primers G1 (59CCCAGGCAACCATTAATCAG-39), G3 (59ATGCCGTTGTTGACGTTGT A-39) and T4 (59GGCTGAAAGGGACGAGTATG-39). Primers T2 and T4 were specific to the 59 and 39 flanking sequences, respectively, of the transgene and could therefore detect the transgene and endogenous genomic DNA simultaneously. The products were amplified with a GeneAmp PCR System 9700 (AB, USA), with cycling parameters of 94uC for 5 minute, 30 cycles of 94uC for 30 seconds, 58uC for 30 seconds and 72uC for 1 minute, followed by a final extension at 72uC for 5 minutes.the chromosome slides were incubated at 65uC for 2 h. Trypsin treatment was performed with 0.05 trypsin in PBS for 13 seconds, and then the slides were stained with 10 Giemsa (Gibco, USA) for 8 minutes. The metaphases with the best pattern were photographed with an Olympus BX53 microscope, and the karyotype was analyzed with Karyo 3.1 software. Chromosomal integration of the transgene was demonstrated by FISH after GTG-binding. Alexa-dUTP was incorporated into a probe containing the entire linearized hLF BAC construct using the BioPrime DNA Labeling System (Invitrogen, USA), which could.
