Skip to main content
. 2022 Feb 4;11:e70207. doi: 10.7554/eLife.70207

Figure 3. Nnt deletion in 6J but not Gzma-/- mice.

(a) Whole-genome sequencing (WGS) of Gzma-/- mice aligned to the 6J (MM10) reference genome build, illustrating the insertion site of the neomycin cassette into the Gzma gene to create the knockout. (b) As for (a) but showing the site of the Nnt deletion, with the additional 12 nucleotides present in the 6J genome. (c) WGS of Gzma-/- and 6J mice aligned to the C3H/HeJ reference genome, illustrating that the Nnt deletion present in 6J mice is absent in Gzma-/- mice. The deletion is in chromosome 13; position 120,695,141–120,711,874 (C3H/HeJ numbering). (d) Reads from RNA-Seq of chikungunya virus (CHIKV)-infected 6J and Gzma-/- mice aligned to the C3H/HeJ and 6J (MM10) reference genomes showing the Sashimi plot (Integrative Genomics Viewer) for the Nnt gene. (e) RT-PCR of testes using primers either side of exons 8–12 in the Nnt mRNA.

Figure 3.

Figure 3—figure supplement 1. Gzma-/- mice have a mixed 6N (yellow)/6J (green) background.

Figure 3—figure supplement 1.

(a) Single-nucleotide polymorphisms (Simon, 2013). (b) Single-nucleotide polymorphisms (Mekada et al., 2015). (c) Insertions/deletions (Simon, 2013). Whole-genome sequencing (WGS) data for Gzma-/- mice provided in PRJNA664888. Nnt highlighted in red box.
Figure 3—figure supplement 2. Nnt and Gzma gene locations and backcrossing requirements.

Figure 3—figure supplement 2.

(a) Both Gzma and Nnt genes are located on mouse chromosome 13 and are separated by about 6.2 million bp. (b) Over 30 backcrosses would be needed to separate the Gzma locus from the Nnt locus (graph taken from http://www.informatics.jax.org/silver/index.shtml).
Figure 3—figure supplement 3. Nnt locus inserts and alignments.

Figure 3—figure supplement 3.

(a) 6J whole-genome sequencing (WGS) reads (SRR8573456) aligned to 6J reference compared with 6N WGS reads (SRR7592164, ERR2814681) aligned to 6J reference, across the 12 nucleotide insert. (b) Proposed source of the 12 nucleotide insertion shown in (a) and Figure 3b. The insertion sequence matches the reverse complement of a sequence ≈1 kb downstream of the Nnt deletion site. The insertion is present in 6J genomes. (c) Location of Nnt exons in the C3H/HeJ mouse genome build (GenBank assembly accession: GCA_001632575.1). Exons highlighted in yellow are missing in 6J mice. (d) A series of WGS with 6J listed as the mouse strain were analyzed as described in Figure 3c and the absence of exon 8–12 confirmed (Y). WGS from CBA/J F1 hybrids (Korablev et al., 2020), mice with an NMRI background (Tumiati et al., 2015), and a WGS with no mouse strain provided, all contained full-length Nnt genes (N).