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1.
Genesis ; 48(5): 328-42, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20213691

RESUMO

The four highly homologous members of the C-terminal EH domain-containing (EHD) protein family (EHD1-4) regulate endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4(-/-) mice confirmed EHD4 deletion. Ehd4(-/-) mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (Day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (Day 31) were seen in the Ehd4(-/-) testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium, and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4(-/-) males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and EHD4 levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age-dependent manner. We conclude that high levels of EHD1 in the adult Ehd4(-/-) testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of postmitotic and postmeiotic germ cells and implicate EHD protein-mediated endocytic recycling as an important process in germ cell development and testis function.


Assuntos
Proteínas de Ligação a DNA/genética , Fertilidade/genética , Proteínas Nucleares/genética , Testículo/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Feminino , Fertilidade/fisiologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Tamanho do Órgão , Contagem de Espermatozoides , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermátides/citologia , Espermátides/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Fatores de Tempo
2.
Transgenic Res ; 19(3): 499-509, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19821046

RESUMO

Using ES cell-mediated transgenesis, we generated a novel mouse strain that permits a temporally and spatially controlled expression of responder genes in embryonic and multiple adult tissues. The transgene was constructed in a way that a CMV enhancer linked to the chicken beta-actin promoter (CAG) drives the expression of the tetracycline-controlled transactivator (tTA) in particular tissues upon Cre-mediated excision of a floxed betageo marker located between the promoter and the tTA. Based on the enzymatic activity of lacZ, the CAG-betageo-tTA construct exhibits a widespread expression and appears to be very strong in the brain, heart, muscle, pancreas, and skin. Like the embryonic stem cell line that was used to generate this strain, the CAG-betageo-tTA transgene is already highly active in preimplantation embryos. Using in vivo bioluminescence imaging on MMTV-Cre, CAG-betageo-tTA, TetO-Luciferase triple transgenic mice and their controls, we demonstrated that the expression of the tTA, which is strictly dependent on the presence of Cre recombinase, induces the activation of the reporter transgene in the absence of any ligands. The tTA-mediated transactivation can be completely ablated through administration of doxycycline, and its subsequent withdrawal lifts the transcriptional block. Based on these characteristics, this novel strain may be useful in experiments that require a sustained expression of transgenes in particular cell types over a prolonged period followed by a rapid downregulation, for example in studies that examine the therapeutic value of cancer-initiating oncogenes during disease progression.


Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica/genética , Camundongos Transgênicos/genética , Transgenes/genética , Actinas/genética , Animais , Southern Blotting , Linhagem Celular , Clonagem Molecular , Citomegalovirus/genética , Primers do DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Galactosídeos , Genótipo , Humanos , Indóis , Luciferases , Camundongos , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes/fisiologia
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