The targeting construct was introduced into RW ES cells by electroporation, followed by G and gancyclov ir double selection.Homologous recombination targeting events were identified in seven of ES cell colon ies. Exons are shown as black boxes and numbered.Directions of the neo and tk genes are depicted by arrows.The wildtype fragment is kb, and the mutant one is about kb.Northern blot analyses of RNA isolated from wildtype and null mouse lung and heart tissues.Overview of the skeletal system was obtained by stain ing cleared specimens of the mouse skeletonwith A lizarin red S and A lcian blue.For histochemical or immunohistological stain ing, tissues were fixed in neutral buf fered formalin and decalcified in either EDTA or formic acid.Specific probes were generated by PCR so that sense primers wereflanked by a SP promoter sequence and antisense primer wasflanked by T promoter sequence.Brief ly, tissues were homogen ized in cold cell lysis buf fer contain ing a protease inhibitor mixture.The lysate was incubated on ice for min and centrifuged, rpm for min at C.Growth chart of y and y mice demonstrates dramatic retardation in the growth of the y mice, showing a decrease in body weight during the last days of life.Af ter Dasatinib implantation, erythromycinyophthalmic ointment was applied to each eye.The corneal neovascularization was examined by a slitlamp biomicroscope on day af ter pellet implantation.Heterozygous mice were morphologically indistinguishable from thewildtype littermates, and they had comparable weight, grow th rates, and external appearance. In contrast, null mice could be distinguished at birth fromwildtype littermates by a slightly domed shape of the skull and a short snout, as well as byvisually prominent suture lines in the cran ium.It was most marked immediately before death of null mice around wk of age, when they only had of the body weight ofwildtype mice. From wk af ter birth, the null mice were less active and had lax sk in.They suf fered from wasting and could not be successfully weaned.A ll bony elements in the appendicular and ax ial skeletons of the null mice were proportionately reduced in size relative towildtype and heterozygous mice. Thus, the bones were markedly shorter and thinner, but otherw ise they displayed normal anatomical features.Highresolution digital radiography and computerized tomography of the heads of wkoldwildtype and null mice revealed that the null mice have a sign ificantly shorter cran iofacial skeleton.This is because of shorten ing of the mandible and max illa and the bones of the base of the skull. Upper and lower incisors are short, and the upper incisor is cur ved inward, resulting in malocclusion.The cran ial cav ity is sign ificantly smaller and the vertex region shows no mineralization.CT scans emphasized the relative lack of mineralization in the null mice. The enlargement of the hypertrophic zones was subsequentlyvisible, but incrementally smaller throughout postnatal life.Note the disorganized Clofazimine structure and low cell number in the proliferative zone in y mice.The lack of vascularization and bone formation in the epiphyseal secondary ossication center of mutant mice can also be observed.Comparison of secondary ossication center of the distal femur from dayold mice.