Fig. 3.

Correct sequence of E64D and E64G mRNAs. RNA from ES cell clones that yielded E64D and E64G mice was reverse transcribed and amplified by PCR with primers based on the reference sequence for mouse Aα mRNA, NM_016891.3 (top line, wild type [WT]). Nucleotide (nt) numbers are given in the right column. Only relevant sequence stretches are shown. The forward primer (nt 24 to 44) binds 5′ of the ATG start codon (nt 53 to 55), the reverse primer (nt 2047 to 2026) 3′ of the TGA stop codon (nt 1820 to 1822). The PCR products were cloned, and clones sensitive to NheI (GCTAGC at nt 250 to 255) were sequenced. The obtained sequences showed codon 64 as GAC for E64D and as GGC for E64G (nt 242 to 244). Stars indicate identity. Silent substitutions (nt 247 to 259) 3′ of E64D and E64G permitted design of primers that distinguish WT (primer <E), E64D (<D), and E64G (<G). The sequence of E64D mRNA from liver and kidney of E64D mice was also correct.