Fig. 1.
Generation and molecular characterization of EA2 knockin mice. (A) The EA2 knockin construct contained a c.4486T>G substitution, coding a p.F1406C missense in exon 26 of Cacna1a and a neomycin resistance cassette (neor), flanked by two loxP sites, upstream of exon 27. The targeting construct was introduced into C57BL/6J–derived ES cells via homologous recombination and cells carrying the construct were injected into blastocysts. (B) A series of PCRs from the DNA of F1 mice were used to verify correct homologous recombination. Reactions from primers P1 and P3 illustrate proper insertion of the 5’ end of the construct and reactions from primers P4 and P6 verify 3’ recombination. (C) DNA and mRNA sequences illustrate the presence of the c.4486T>G substitution in the PCR product of genomic DNA and RT-PCR product of brain mRNA from +/+ and EA2/EA2 mice. (D) Western blot shows the normal expression of CaV2.1 in frontal cortex and cerebellum.