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[求助]
求助翻譯 關(guān)于HIF和shRNA
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求助翻譯： 菜鳥一個(gè)，這段有點(diǎn)搞不懂作者的意圖是什么。。。 To investigate the role of each of the HIF-a subunits in human HSPCs, we constructed bicistronic lentiviral vectors carrying two distinct promoters, EF1a and H1, driving green fluorescent pro- tein (GFP) (reporter) and a small hairpin RNA (shRNA) against the HIF-1a (sequence published in Rezvani et al., 2007) or HIF-2a subunit, respectively (Figure S1B). To confirm the specificity and efficiency of these vectors, CD34+ cells were transduced (45%–80% transduction efficiency), FACS-sorted 4 days post- transduction, and cultured for an additional 4 days prior to our assessment of mRNA levels. A significant reduction in mRNA levels was observed for both HIF-1a and HIF-2a vectors relative to controls, with a decrease of 88% and 78%, respectively (Fig- ure 1B). The KD was also confirmed at the protein level in CD34+ cells (Figure 1C) and in 293T cells (Figure S1C). The shHIF1a used in this study has been previously validated elsewhere (Rezvani et al., 2007). However, to confirm the specificity and exclude off-target effects of the shRNA for HIF-2a, we tested a second shHIF2a (shHIF2a.2) (Figure S1D). Using this second vector, we confirmed an efficient KD at the protein level, as well as similar defects in vivo compared to the first shRNA used (shHIF2a.1) (Figure S1E). We then used the construct that gave us the best silencing activity (shHIF2a.1) for the rest of this study. 還有構(gòu)建的shRNA作用是不是就是表達(dá)HIF1a和HIF2a？ 下游靶基因的表達(dá)減少是否意味著HIF1a和HIF2a的表達(dá)減少？ |
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