Figure 2. Membralin transgene [Tg (membralin)] rescues GluN3B/membralin (DKO) mice.
(A) Generation of the transgenic constructs expressing membralin. The murine prion promoter was cloned with full-length mouse membralin cDNA followed by the IRES and the EGFP sequence. The entire transgenic sequence was isolated by enzymatic digestion and used to generate Tg (membralin) mice. (B) DKO/Tg (membralin) mice were viable and fertile. Left: genotyping by PCR showed that 3 littermates (#3, 4 and 5) from a breeding of DKO and Tg (membralin) mice were positive to DKO primers and negative to WT primers (same primers used as in Figure 1). Of the three DKO mice, the two receiving membralin transgene (#3 and 5, as positively identified by GFP primers) were rescued. Right: Weight of the 7 littermates was monitored after birth. Only DKO mouse #4, which was negative for the membralin transgene, showed weight loss after P3 and died at P5.5. Data shown here are from one representative litter (5 independent litters were analyzed with similar results). (C) Gene expression analysis by RT-PCR for mice #1, 3, 4 of the same litter as in B. Membralin transgene expression is seen in the brains of littermates #1 and 3, but not 4, whereas GluN3B is only expressed in #1. Membralin and GluN3B are not expressed in muscle samples. GAPDH is expressed in both brain and muscles of all mice and served as a positive control. (D) Rescued DKO/Tg (membralin) mice lived to adulthood without any sign of paresis.
Figure 2—figure supplement 1. Expression of GFP-membralin transgene in spinal cord of membralin transgenic mice.
Figure 2—figure supplement 2. GluN3B transgene cannot rescue GluN3B/membralin DKO mice.
