Figure 4. An eight base-pair deletion in Draxin in BTBR strains truncates and reduces DRAXIN protein expression.

Exome sequencing of a candidate region on mouse chromosome 4 (A) and confirmatory Sanger sequencing found an eight base-pair deletion in Draxin, which introduces a premature stop codon (B), predicted to truncate DRAXIN protein (B, E). (C) In situ hybridisation against 3′ C57 or BTBR Draxin demonstrates a similar pattern of Draxin mRNA expression in C57 and BTBR parental strains and in the BTBR × C57 N2 mice. Fluorescent immunohistochemistry for DRAXIN protein (bottom-left panels) demonstrates that DRAXIN is highly expressed in C57 mice at E15 but not in BTBR mice. (D) Cell lysates derived from HEK293T cells expressing pCag-eYFP or pCag-iresGFP with either BTBR or C57 Draxin coding sequences were incubated with anti-DRAXIN, anti- β-ACTIN, and anti-GFP antibodies. Specific bands at ~18 kD and ~40–45 kD are shown for DRAXIN (red boxes) and demonstrate that BTBR Draxin produces a protein of reduced molecular weight, indicating truncation. Midline tissue lysates from E15 C57 and BTBR mice incubated with anti-DRAXIN and anti-β-ACTIN antibodies reveal (red boxes) specific bands at ~40–60 kD and ~42 kD, respectively, indicating that DRAXIN expression is severely reduced in BTBR mice.