Fig. 1. Generation of knock-in animals expressing Erbb2 tyrosine phosphorylation mutants. The knock-in targeting vector was constructed such that exon 1 of the mouse Erbb2 gene was replaced with either (A) a rat Erbb2 cDNA or (B) a cDNA encoding a mutant Erbb2 receptor harboring the Y1028F mutation. The cDNA is followed by a PGK-Neomycin/SV40-polyA expression cassette and is targeted to the endogenous Erbb2 locus by homologous 5' and 3' flanking arms, placing it directly under the transcriptional control of the endogenous Erbb2 promoter. The targeted allele introduces an additional HindIII site that was used to distinguish between the endogenous wild-type allele (7.5 kb fragment) and the targeted allele (4.0 kb fragment) in Southern blot analyses (A, inset). (C) Schematic representation of the Erbb2 receptor depicted with the five tyrosine autophosphorylation sites in the C-terminal tail and with the corresponding amino acid number. Note that the numbering of the amino acids is based on the rat Erbb2 sequence and will be referred to herein by these designates. Also shown are the three individual tyrosine-to-phenylalanine mutations described in this report: Y1028F, Y1144F and Y1227F. (D) Alignment of the amino acid sequences surrounding tyrosine 1028 in Erbb2 and tyrosine 992 in Egfr. (E) Expression analysis of the knock-in allele. Total protein lysates prepared from E12.5 wild-type and Erbb2 cDNA knock-in embryos were used to detect Erbb2 levels. Detection of Grb2 protein (lower panel) served as an internal loading control.