RESUMO
Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.
Assuntos
Vetores Genéticos , Lentivirus/genética , Glicoproteínas de Membrana/genética , Transdução Genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular , Glicosilação , Humanos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/química , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Especificidade da Espécie , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/química , Proteínas Virais/análise , Proteínas Virais/química , Proteínas Virais/genética , Vírion/químicaRESUMO
OBJECTIVE: Neurological injury associated with present day labor and delivery is thought to be unusual. The purpose of this study was to estimate the incidence, severity, and duration of postpartum lumbosacral spine and lower extremity nerve injury and identify factors related to nerve injury. METHODS: All women who delivered a live-born infant from July 1997 through June 1998 were asked about symptoms of lumbosacral spine and lower extremity nerve injury the day after delivery. Women with symptoms were examined by a physiatrist to confirm injury, and their cases were then followed by telephone until the symptoms resolved. Maternal variables (including prospective documentation of time spent pushing in various positions) and fetal variables that might be associated with risk of nerve injury were compared between women with injury and those without. RESULTS: Six thousand fifty-seven women delivered live-born infants; 6,048 were interviewed and 56 had a confirmed new nerve injury, an incidence of 0.92%. Factors found by logistic regression analysis to be associated with nerve injury were nulliparity and prolonged second stage of labor. Women with nerve injury spent more time pushing in the semi-Fowler-lithotomy position than women without injury. The median duration of symptoms was 2 months. CONCLUSION: The estimated incidence of postpartum nerve injury was greater than reported from previous studies and is associated with nulliparity and prolonged second stage of labor.