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. 2015 May 21;5:10482. doi: 10.1038/srep10482

Figure 2.

Figure 2

Targeted replacement of the hLF gene with the BLG locus in goat fetal fibroblasts. (A) Schematic overview of the knock-in strategy for the BLG locus. (top) Schematic representation of the BLG locus and the targeting vector with the hLF cDNA and bovine growth hormone polyadenylation signals. (bottom) Schematic of the targeted integration of hLF gene and PGK-neo cassette. (B) Junction PCR analysis for screening the targeting events by using donor pBLG-hLF-neo. The positions of primers for the junction PCRs are indicated on the knock-in locus schematic. Correctly targeted clones are in red. (top) Analysis of drug-resistant clones by 3′ junction PCR with primers LP3 and LP4. A representative panel of 10 clones showed the expected 1.9 kb band. (bottom) Genomic DNA of the 3′ junction PCR-positive clones was PCR-amplified with primers LP1 and LP2. A representative panel of 10 clones showed the expected 2.7 kb band. (C) Southern blot analysis of BLG+/− goats. Genomic DNA was digested with BamHI and hybridized with the external 3′ probe or the internal neo probe. The 3′ probe detects a 4.5 kb band for the wild-type and an 8.8 kb band for the hLF targeted integration. The neo probe detects the unique 8.8 kb band for the targeted integration. (D) Sequence comparison of the 5′ and 3′ junctions between the wild-type genomic DNA and the donor DNA of a representative BLG+/hLF goat (#L2). Restriction sites in the 5′ and 3′ ends of the donor DNA are underlined. The 5′ and 3′ ends of the expression cassette are colored gray; the loxP sites are identical to those in Fig. 1E. Letters in boldface indicate the 16 bases of the BLG gene that were replaced by the hLF gene and the PGK-neo cassette. “sig” indicates the signal peptide sequence of the BLG gene.