RESUMO
The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.
Assuntos
Proteínas de Membrana/fisiologia , Doenças Urológicas/metabolismo , Urotélio/fisiologia , Animais , Predisposição Genética para Doença , Nefropatias/genética , Nefropatias/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fenótipo , Doenças Urológicas/genética , Doenças Urológicas/patologia , Uroplaquina II , Uroplaquina III , Urotélio/citologia , Urotélio/patologia , Refluxo Vesicoureteral/metabolismoRESUMO
Successful experiments involving the production of transgenic mice by pronuclear microinjection are currently limited by low efficiency of random transgene integration into the mouse genome. Furthermore, not all transgenic mice express integrated transgenes, or in other words are effective as functional transgenic mice expressing the desired product of the transgene, thus allowing accomplishment of the ultimate experimental goal--in vivo analysis of the function of the gene or gene network. The purpose of this review is to look at the current state of transgenic technology, utilizing a pronuclear microinjection method as the most accepted way of gene transfer into the mouse genome.
Assuntos
Núcleo Celular/genética , DNA/genética , Técnicas de Transferência de Genes , Camundongos Transgênicos/fisiologia , Microinjeções/métodos , Transgenes , Animais , Animais Geneticamente Modificados/genética , DNA/administração & dosagem , Modelos Animais de Doenças , Genoma , Humanos , Camundongos , Camundongos Transgênicos/genética , Plasmídeos/genéticaRESUMO
Transgenic mouse production via pronuclear microinjection is a complex process consisting of a number of sequential steps. Many different factors contribute to the effectiveness of each step and thus influence the overall efficiency of transgenic mouse production. The response of egg donor females to superovulation, the fertilization rate, egg survival after injection, ability of manipulated embryos to implant and develop to term, and concentration and purity of the injected DNA all contribute to transgenic production efficiency. We evaluated and compared the efficiency of transgenic mouse production using four different egg donor mouse strains: B6D2/F1 hybrids, Swiss Webster (SW) outbred, and inbred FVB/N and C57BL/6. The data included experiments involving approximately 350 DNA transgene constructs performed by a high capacity core transgenic mouse facility. Significant influences of particular genetic backgrounds on the efficiency of different steps of the production process were found. Except for egg production, FVB/N mice consistently produced the highest efficiency of transgenic mouse production at each step of the process. B6D2/F2 hybrid eggs are also quite efficient, but lyze more frequently than FVB/N eggs after DNA microinjection. SW eggs on the other hand block at the 1-cell stage more often than eggs from the other strains. Finally, using C57BL/6 eggs the main limiting factor is that the fetuses derived from injected eggs do not develop to term as often as the other strains. Based on our studies, the procedure for transgenic mouse production can be modified for each egg donor strain in order to overcome any deficiencies, and thus to increase the overall efficiency of transgenic mouse production.