RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most challenging infectious disease of pig populations causing devastating economic loss to swine industry. Reverse genetics allow to engineer modified viruses such attenuated strains for vaccine development. Some reverse genetic systems were described for PRRSVs but, due to genome complexity of PRRSVs, construction and modification of such systems remain laborious and time-consuming. In this study, we described a reverse genetics approach based on the "Infectious-Subgenomic Amplicons" (ISA) method to rescue infectious PRRSV particles. Permissive cells were transfected with 4 overlapping synthetic DNA fragments covering the entire genome of PRRSV which allowed the rapid reconstruction of the complete virus genome and the subsequent generation of infectious wild-type particles within days. The ISA method represent a rapid alternative of conventional reverse genetic systems. This method will help to generate genetically modified and attenuated strains for the development of sanitary countermeasures in the future.
RESUMO
p63, a member of the p53 family, is essential for skin morphogenesis and epithelial stem cell maintenance. Here, we report an unexpected role of TAp63 in cardiogenesis. p63 null mice exhibit severe defects in embryonic cardiac development, including dilation of both ventricles, a defect in trabeculation and abnormal septation. This was accompanied by myofibrillar disarray, mitochondrial disorganization, and reduction in spontaneous calcium spikes. By the use of embryonic stem cells (ESCs), we show that TAp63 deficiency prevents expression of pivotal cardiac genes and production of cardiomyocytes. TAp63 is expressed by endodermal cells. Coculture of p63-knockdown ESCs with wild-type ESCs, supplementation with Activin A, or overexpression of GATA-6 rescue cardiogenesis. Therefore, TAp63 acts in a non-cell-autonomous manner by modulating expression of endodermal factors. Our findings uncover a critical role for p63 in cardiogenesis that could be related to human heart disease.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Coração/embriologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Coração/crescimento & desenvolvimento , Immunoblotting , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fosfoproteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Transativadores/genéticaRESUMO
In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.
Assuntos
Células-Tronco Embrionárias/citologia , Células Epidérmicas , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosfoproteínas/genética , Transativadores/genética , TransfecçãoRESUMO
Embryonic stem (ES) cells can be differentiated into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells were able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1(+) neural precursors to undergo specific apoptosis while inducing epidermal differentiation through DeltaNp63 gene activation. We further demonstrated that DeltaNp63 enhances ES-derived ectodermal cell proliferation and is necessary for epidermal commitment. This unique cellular model further provides a powerful tool for identifying the molecular mechanisms controlling normal skin development and for investigating p63-ectodermal dysplasia human congenital pathologies.
