Generation of fyn-transgenic mice and expression of fyn-transgene. (A) Schematic representation of the transgene construct that is composed of a 8.5-kb fragment, including the CaMKIIα promoter, a hybrid intron consisting of an adenovirus splice donor, and an IgG splice acceptor, an EcoRI–DraI fragment of fynB cDNA, and a simain virus 40 poly(A) signal sequences. Transgenic mice were generated as described. The probes for detection of transgene mRNA and for ribonuclease protection assay are indicated with thick lines (probes 1 and 2, respectively). (B) Northern blot analysis on forebrain. Total RNA prepared from wild-type (wt) and transgenic littermates (Tg) was electrophoresed on formaldehyde agarose gel, transferred onto nylon membrane, and hybridized with probe 1 shown in A Upper and fynB cDNA A Lower. (C) Detection of the transgene mRNA in brain sections. Sagittal brain sections prepared from wild-type (wt) and transgenic (Tg) littermates were hybridized with digoxygenin-labeled cRNA probe 2 shown in A. (D) Developmental change in the level of transgene mRNA. Ribonuclease protection assay was performed on 1-day-old (P₁ Tg), 10-day-old (P₁₀ Tg), 3-week-old (P₂₁ Tg) transgenic, adult wild-type (Adult wt), and adult fyn-deficient (Adult fyn⁻) forebrains. The protected bands corresponding to endogenous fyn mRNA (fyn) and the transgene transcript (fyn Tg) are designated by arrows.

Fyn protein, kinase activity, and tyrosine phosphorylation in the fyn-rescue mice. (A) The extracts prepared from forebrains of 2- to 3-month-old wild-type (wt), fyn-deficient (fyn⁻) and fyn-rescue (fyn-rescue) mice were separated on a SDS/10% polyacrylamide gel and blotted with an anti-Fyn antibody. The band corresponding to Fyn protein is designated by an arrow. (B) Proteins from the same extracts were immunoprecipitated with an anti-Fyn antibody conjugated with protein G–Sepharose. The precipitates were incubated with [γ-³²P]ATP in the presence of enolase, and then resolved on a SDS/10% polyacrylamide gel. Fyn and enolase are indicated by arrows. (C) Proteins from the same brain extracts were blotted with an anti-phosphotyrosine antibody, PT66. The arrowheads indicate the bands that show less phosphorylation in fyn-deficient mice.

Hippocampal morphology in fyn-deficient and fyn-rescue mice. (A) Nissl staining of rostral and caudal coronal hippocampal sections of wild-type (wt), fyn-deficient (fyn), and fyn-rescue (rescue) mice. Characteristic abnormal arrangements (arrowheads) of rostral dentate gyrus (DG) and caudal CA3 region in fyn-deficient mice were also observed in the rescue mice. (Bars = 500 μm.) (B) Dendritic organization of the CA1 field in rescue mice. Apical dendrites of the pyramidal cells were revealed by immunohistochemical staining using anti-MAP2 antibody. CA1 pyramidal cell layer (pc) was less tightly packed and ectopic pyramidal cells (arrowheads) were frequently observed in fyn-deficient (fyn) and fyn-rescue (rescue) mice. Dendrites ran across the stratum radiatum (sr) and lacunosum-moleculare (lm), but their lengths were shorter than in wild-type litter mates. (Bar = 50 μm.)

LTP in fyn-rescue and young fyn-deficient mice and Src compensation in fyn-deficient mice. (A) Field EPSP recorded before and after tetanic stimulation in slices from adult wild-type (wt, ▪), fyn-deficient (fyn⁻, ▵), and fyn-rescue mice (fyn-rescue, ▴). Each point is the mean ± SEM of the field EPSP slope. Wild-type mice: n = 8 animals, 13 slices; fyn-deficient mice: n = 9 animals, 16 slices; fyn-rescue mice: n = 11 animals, 32 slices. (B) LTP measured 1 hr after tetanic stimulation. Wild-type (wt) mice: 148.3 ± 8.7% (n = 8); fyn-rescue (fyn-rescue) mice: 135.0 ± 7.2% (n = 9); fyn-deficient (fyn⁻) mice: 112.7 ± 5.7% (n = 11). Wild-type mice versus fyn-rescue mice: t = 1.14, not significant; fyn-rescue mice versus fyn-deficient mice: t = 2.39, P < 0.05. (C) Time course of LTP in wild-type (wt, •) and fyn-deficient (fyn⁻, □) mice. Each point is the percentage of LTP obtained at different age. Wild-type mice: 2 weeks, n = 5; 6 weeks, n = 6; 10 weeks, n = 6; 14 weeks, n = 9. fyn-deficient mice: 2 weeks, n = 12; 6 weeks, n = 7; 10 weeks, n = 4, 14 weeks, n = 6. (D) Src levels in wild-type (wt) and fyn-deficient (fyn) mice of different postnatal ages. Protein extract from mice aged 2, 6, 10, 12, and 14 weeks. The levels of Src are indicated by the arrow.