Hsf4 knockout results in lens abnormalities and developmental defects, notably swollen and loose fiber structure. (A) The wild-type Hsf4 locus, the targeting vector, and the allele following homologous recombination are shown. The targeting vector was used to replace exons 3–5, encoding the DNA-binding domain, with a neomycin resistance gene. (B) Analysis of genomic DNA, cDNA, and protein of the Hsf4 gene from Hsf4 knockout mouse by PCR, RT-RCR, and Western blotting analysis. PCR primers flanking the targeted exons amplified 2.6-kb and 1.6-kb bands in Hsf4-/- and Hsf4+/+ mice, respectively. Loss of wild-type expression of the Hsf4 gene in Hsf4-/- mice was confirmed by the lack of a PCR product after reverse transcription-PCR of total RNA with one of primers within the targeted region. Western blot analysis of lens extracts of 8-week-old mice using a specific antibody against mouse Hsf4 protein showed that Hsf4 protein was absent in Hsf4-/- mice. (C) Slit-lamp images of mouse lens and histological examination of lens nuclear region sections of 8-week-old mice. Arrows indicate normal and loose fibers in lens nuclear regions of Hsf4+/+, Hsf4+/-, and Hsf4-/- mice. Bar, 50 um. (D) SEM images showing the loose and abnormal fibers of Hsf4 knockout mice compared with the normal structures of wild-type mice. Bar, 500 um above and 5 um below.