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. 2014 Nov 5;43(1):e5. doi: 10.1093/nar/gku1050

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — Application of snoVectors to miRNA expression and the selection of stable cell lines. (A) Expression of pre-miRNA-let-7g from the snoVector. Left, transfection of pZW1-snoVectors and the processing of transcribed pre-RNAs from transfected plasmids were indicated by EGFP fluorescence intensity. Right, NB revealed that pre-let-7g is expressed from pZW1-snoVector with total RNAs collected from HeLa cells transfected with the indicated plasmid. Transcripts of expressed RNAs were probed with Dig-labeled antisense SNORD116-13. NT, no transfection; EV, transfection with an empty vector. (B) Let-7g was produced from the pZW1-snoVector transfection revealed in (A). The production of let-7g was measured by NB with a [γ-32P] ATP labeled DNA probe complementary to the mature let-7g. The loading control is 5.8S rRNA. (C) Stable cell line selection with Kan/Neo resistance. Note that snoRNA-ended mNEAT1-v3 was stably produced in PA1 cells as revealed by NB and was predominately retained in the nucleus at the passage 20 (P20) after obtained the stable cells. (D) The stable cell line expressing snoRNA-ended mNEAT1-v3 (green, by RNA ISH) at similar levels in individual cells at different passages P20 (top) and P30 (bottom).