The relative luciferase activity normalized to the value of pRL SV40 activity.

IFA showed that NSPC iPSCs expressed the NSC markers Nestin, Sox2, and Pax6, indicating that NSPC iPSCs have the authentic NSPC phenotype. In vitro HCMV infection of iPSC derived NSPCs To examine the susceptibility of NSPC iPSCs to HCMV in fection, these cells were inoculated in vitro with the HCMV Towne strain at an MOI of ABT-888 臨床試験 1 PFU per cell. On the second day post infection, NSPC iPSCs started to show morphological changes including increased cell vol ume and cell fusion, and the number of cells with these changes increased until 7 dpi. To examine whether NSPC iPSCs were capable of supporting HCMV gene expression, total RNA extracted from the infected NSPC iPSCs was analyzed by RT PCR. As shown in Figure 2B, mRNAs encoding IE1, IE2, vIL 10, and pp65 as well as those encoding HCMV anti apoptotic proteins, such as UL36 and UL38, were detected.<br><br> The kinetics of HCMV gene expression was analyzed by quantitative real time RT PCR. IE1 mRNA was detected first on 1 dpi and increased steadily until 5 dpi. mRNAs for UL89 and UL136 were detected somewhat later and increased grad ually until 7 dpi. The results showed the NSPC iPSCs are susceptible to HCMV infection and allow the expression of several オーダー Afatinib viral genes of both early and late functions. Expression of HCMV genes in NSPC iPSCs was evaluated at the protein level by immunoblot analysis on day 1, 2, 5, and 7 following HCMV infection. As shown in Figure 2D, the immediate early protein IE1 was first detected at 1 dpi and its level increased until 5 dpi. Another immediate early protein IE2 was detected a little later, becoming visible at 5 dpi.<br><br> The expression of the HCMV lower matrix protein pp65, already visible at 1 dpi, was markedly ele vated at 5 and 7 dpi. The HCMV envelope glycoprotein B was detected at 5 to 7 dpi. Thus the expression of HCMV proteins 価格 AG-1478 of both immediate early and late func tions was demonstrated in NSPC iPSCs. We next examined the expression of cellular mRNAs encoding the pluripotency and neural differentiation markers. Expression of the iPSC markers Nanog and Oct 4 remained at low levels following infec tion with HCMV, although that of Nanog tapered. While expression of the NSPC markers Sox2 and Pax6 were kept at high levels following HCMV infection, that of another NSPC marker Nestin was markedly suppressed at 7 dpi.<br><br> In addition, expression of the neuronal marker microtubule associated protein 2, the astrocyte marker glial fibrillary acidic protein, and the oligodendrocyte marker oligodendrocyte specific protein was detected at low levels. Interestingly, Sox1, a marker specific to the neuroectodermal lineages, was markedly upregulated following infection with HCMV. Expression of the NSPC markers was evaluated also at the protein level by immunoblot analysis on 1, 2, 5, and 7 dpi. In accordance with the results with RT PCR, expression of Pax6 and Nestin was confirmed, and that of Nestin was found markedly decreased 7 dpi. Another NSPC marker Musashi 1 was also detected. To examine whether HCMV infected NSPC iPSCs produce infectious virions, culture supernatants collected from Towne HCMV infected NSPC iPSCs were inoculated to hTERT BJ1 cells. Inoculated cells expressed IE1 IE2 indicating that infectious virions were produced from HCMV infected NSPC iPSCs.