Expected. Similarly, the forward primer T4 from the transgene with G

Expected. Similarly, the forward primer T4 from the transgene with G3 generates a 422-bp product when the transgene is present in the 39 flanking transgene locus, which will generate a 633-bp product from wildtype cattle and 633- and 422-bp products in the purchase JW-74 transgenic cattle. All samples analyzed by PCR exhibited the same breakpoint that had been identified by next-generation sequencing, suggesting that the transgene is stable between generations (Figure 3B and Figure S1). Furthermore, the rearrangement of genomic DNA, including deletion or translocation, has been observed at the integration sites of transgenes in previous studies [21,22]. A deletion of an 11nucleotide portion of the cow genome at the insertion site was also observed (67515636?7515646 of chromosome 15), which exhibited a characteristic signature of transgene integration (Figure 2).Verification of Transgene Chromosomal Location by FISHGTG-banding was performed on metaphase spreads of fibroblast cells from the transgenic cattle, and more thanReliable Method for Transgene IdentificationFigure 6. Schematic representation of the BAC rearrangements in transgenes. The positions of the junctions between the hLF BAC fragment (gray box) and the pBeloBAC vector (open box) are indicated, with arrowheads for orientation. An 85-bp unknown sequence was identified in the Type A molecule (hatched box). All six of these configurations should have been concatenated in an unknown format at the transgene integration site. doi:10.1371/journal.pone.0050348.gmetaphase spreads were acquired from each animal. The banded metaphases were identified and photographed before hybridization, and the same metaphases were photographed again after hybridization to detect the signals. A large number of metaphase spreads must be observed because not all will display hybridization signals. In this study, approximately 50 of the metaphase spreads exhibited positive FISH signals, indicating the presence of the transgene. Next, 20 metaphase spreads that exhibited both clear GTG-banding patterns and positive FISH signals were used to confirm the identity of the signal-bearing chromosome according to the standard cattle karyotype proposed by ISCNDB 2000 [23]. As expected, positive FISH signals were observed on chromosome 15 from the DNA of #040825 (Figure 4) as well as from the DNA of #050211 and #101026 (Figures S2 and S3). These results confirm that the transgene had a single integration site on chromosome 15q26, in agreement with the next-generation sequencing results.Determination of Transgene Rearrangement and Copy NumberAlthough the sequencing coverage of the cow genome was approximately 106for each DNA sample, the effective sequencing depth of the hLF BAC ranged from 206 to 506 (Figure 5). This discrepancy implied that multiple copies of 1527786 the hLF BAC had been incorporated into the cow genome and that some copies might be incomplete. This conclusion was supported by quantitative PCR, which revealed a variable copy number in different regions of Solvent Yellow 14 web hLFBAC, from 2 to 8 (data not shown). In addition, to quantify the transgene copy number, all incidences of an abnormal paired-end read that bridged the BACs and the pBeloBAC vector were analyzed, and a complex pattern of sequencing depth distribution of the pBeloBAC vector was observed (Figure 6), suggesting that a complex rearrangement of transgenes may have occurred upon integration. Overall, six different BAC-vector junctions were identified in the transgenic cat.Expected. Similarly, the forward primer T4 from the transgene with G3 generates a 422-bp product when the transgene is present in the 39 flanking transgene locus, which will generate a 633-bp product from wildtype cattle and 633- and 422-bp products in the transgenic cattle. All samples analyzed by PCR exhibited the same breakpoint that had been identified by next-generation sequencing, suggesting that the transgene is stable between generations (Figure 3B and Figure S1). Furthermore, the rearrangement of genomic DNA, including deletion or translocation, has been observed at the integration sites of transgenes in previous studies [21,22]. A deletion of an 11nucleotide portion of the cow genome at the insertion site was also observed (67515636?7515646 of chromosome 15), which exhibited a characteristic signature of transgene integration (Figure 2).Verification of Transgene Chromosomal Location by FISHGTG-banding was performed on metaphase spreads of fibroblast cells from the transgenic cattle, and more thanReliable Method for Transgene IdentificationFigure 6. Schematic representation of the BAC rearrangements in transgenes. The positions of the junctions between the hLF BAC fragment (gray box) and the pBeloBAC vector (open box) are indicated, with arrowheads for orientation. An 85-bp unknown sequence was identified in the Type A molecule (hatched box). All six of these configurations should have been concatenated in an unknown format at the transgene integration site. doi:10.1371/journal.pone.0050348.gmetaphase spreads were acquired from each animal. The banded metaphases were identified and photographed before hybridization, and the same metaphases were photographed again after hybridization to detect the signals. A large number of metaphase spreads must be observed because not all will display hybridization signals. In this study, approximately 50 of the metaphase spreads exhibited positive FISH signals, indicating the presence of the transgene. Next, 20 metaphase spreads that exhibited both clear GTG-banding patterns and positive FISH signals were used to confirm the identity of the signal-bearing chromosome according to the standard cattle karyotype proposed by ISCNDB 2000 [23]. As expected, positive FISH signals were observed on chromosome 15 from the DNA of #040825 (Figure 4) as well as from the DNA of #050211 and #101026 (Figures S2 and S3). These results confirm that the transgene had a single integration site on chromosome 15q26, in agreement with the next-generation sequencing results.Determination of Transgene Rearrangement and Copy NumberAlthough the sequencing coverage of the cow genome was approximately 106for each DNA sample, the effective sequencing depth of the hLF BAC ranged from 206 to 506 (Figure 5). This discrepancy implied that multiple copies of 1527786 the hLF BAC had been incorporated into the cow genome and that some copies might be incomplete. This conclusion was supported by quantitative PCR, which revealed a variable copy number in different regions of hLFBAC, from 2 to 8 (data not shown). In addition, to quantify the transgene copy number, all incidences of an abnormal paired-end read that bridged the BACs and the pBeloBAC vector were analyzed, and a complex pattern of sequencing depth distribution of the pBeloBAC vector was observed (Figure 6), suggesting that a complex rearrangement of transgenes may have occurred upon integration. Overall, six different BAC-vector junctions were identified in the transgenic cat.