ライフサイクルとヒューマンケア: 高齢者への健康支援(平成23年度教養コア科目) 授業資料ナビゲータ(PathFinder)

担当教員:黒田久美子,野地有子,今村恵美子,永野みどり平成23年度(2011)教養コア科目授業B(こころと発達),授業コード:G14B1410

Primers and probes for qPCR and generation of recombinant viruses.

<p>Primers and probes for qPCR and generation of recombinant viruses.</p

Integration efficiency and genome maintenance of the ΔTMR mutant in the U2OS integration system.

<p><b>(A)</b> Integration frequency was quantified by determining the integration status of at least 90 metaphases. Significant differences between wt and ΔTMR (Mann-Whitney U-test, p < 0.05) are indicated with an asterisk (*). Results are shown as means of three independent experiments with standard errors. Representative metaphase images are shown on the right. Scale bar corresponds to 10μm. <b>(B)</b> 300 interphase nuclei were examined for the presence of HHV-6A. Significant differences between wt and ΔTMR (Mann-Whitney U-test, p < 0.05) are indicated with an asterisk (*). Results are shown as means of three independent experiments with standard errors. Representative interphase images are shown on the right. Scale bar corresponds to 10μm. <b>(C)</b> Maintenance of the HHV-6A genome was determined by qPCR analysis at d0 and d14 post sorting. Copy numbers per 1x 10⁶ cells are shown as means of three independent experiments with corresponding standard errors. Significant differences between wt and ΔTMR (Mann-Whitney U-test, p < 0.01) are indicated with asterisks (**).</p

Generation and characterization of the ΔTMR mutant.

<p><b>(A)</b> Schematic representation of the HHV-6A genome with deletion of the TMR (ΔTMR). <b>(B)</b> RFLP pattern of the wt, the ΔpTMR intermediate and the double deletion mutant ΔTMR upon digestion with <i>Sac</i>I analyzed on a 0.8% agarose gel o/n at 65 V. M = marker. Sizes of the marker fragments are indicated on the left. Red boxes highlight the fragments containing the target regions, where the expected band shifts can be observed. <b>(C)</b> Corresponding southern blot analysis detecting TMR sequences of the impTMR (upper panel) and pTMR (lower panel) after <i>Sac</i>I digestion of the indicated BAC clones using a DIG-labeled TMR probe. <b>(D)</b> Growth kinetics comparing replication properties of wt and ΔTMR mutant virus in JJHan cells. HHV-6A genome copy numbers were detected by qPCR. Copy numbers per 1x 10⁶ cells are shown as means of three independent experiments with standard errors.</p