Figure 1.
Pmel-1 TCR tetramer staining distinguishes the gene dosage of pmel-1 transgene in CD8+ T cells.
Staining of pmel-1 heterozygous or homozygous CD8+ T cells with antibodies recognizing pmel-1 TCR β chain Vβ13 (upper panel) or tetramers recognizing pmel-1 TCR (lower panel).
Figure 2.
This chart depicts the analysis of NGS data from Whole Genome Sequencing. In this analysis, standard tools for small structural variation detection were used to predict large insertions based on short paired-end (2*100 bp) sequence reads. Both SVDetect and DELLY extract data from a reference sequence file and a sequence alignment (BAM) file and generate filtered lists of predicted structural variations, their genomic coordinates, and confidence scores as a tab-separated text. Downstream steps, such as data visualization, cross-sectioning and ranking of SVs were done in the Integrative Genomics Viewer and Excel.
Figure 3.
Read alignments around the insertion site of pmel-1.
An ideogram of chromosome 2 is shown at the top with the red bar indicating the area of insertion. Gray bars represent concordant read pairs. Red and blue colors represent read pairs where the insert size is larger than expected (deletion) or smaller than expected (insertion), respectively. Discordantly mapped reads are coded by the chromosome on which their mates can be found, so reads represented by magenta and aquamarine rectangles have mates that mapped to chromosomes 6 and 14, respectively. Other colors represent genomic “noise”. Multicolored blocks represent misaligned areas within reads. Soft-clipped reads were represented by grey bar with multicolored blocks at the ends. Misaligned bases in soft-clipped reads are shown in blue (C), green (A), red (T) and orange (G) color, respectively. (A) A snap shot of pmel-1 sequence reads aligned to the reference mouse genome near the integration area. Eight magenta-colored discordant reads are on the left side and five aquamarine-colored are on the right side. (B) An enlarged view of pmel-1 sequence reads, which shows five soft-clipped reads, one on right side, four on left sides of the insertion area. (C) A nucleotide level view of pmel-1 reads near the insertion area. This panel shows four soft-clipped reads that mapped partially to the β chain on chromosome 6 and partially to chromosome 2 of the reference genome.
Figure 4.
PCR analysis distinguishes the homozygosity or heterozygosity of pmel-1 transgene.
(A) Diagram of PCR primers designed from the predicted pmel-1 TCR β transgene insertion site on chromosome 2. (B) PCR results of pmel-1 homozygous or heterozygous animals. (C) Conceptual model of tandem integration of the pmel-1 α and β chains in the genome.