Regulation of Emx2 Expression by Antisense Transcripts in Murine Cortico-Cerebral Precursors (original) (raw)
Figure 1
Synopsis of molecular tools used for studying expression and function of Emx2OS.
(A) Representation of the murine Emx2 locus, with the _Emx2_-mRNA and the _Emx2OS_-ncRNA transcription units (red). Genomic localization of the riboprobe used for in situ hybridization analysis of Emx2OS (blue bar). Genomic localization of the oligonucleotides employed for quantitative RT-PCR evaluation of _Emx2OS_-ncRNA (P1 and P3), _Emx2_-mRNA (N2F and N2R) and _Emx2_-pre-mRNA (I1, I2, I3 and I4) (blue arrowheads). (B) Structure of the bi-cistronic plasmids employed for assaying activities of artificial miRNAs against _Emx2OS_-ncRNA in HeLa cells and of lentivectors for overexpressing these miRNAs in primary cells. Localization of miR-α_Emx2_OS-542 and -774 (blue arrowheads) as well as of their responsive element, miR-RE, with respect to the antisense transcript. miR-RE (dark yellow), showed enlarged over the antisense transcript, extends across the 3rd and the 4th exons of it. (C) Structure of the “driver” lentivector, guiding constitutive expression of rtTA2S-M2, and of “expressor” lentivectors, guiding rtTA/doxycycline-dependent expression of ncRNA/IRES/eGFPcds modules. Genomic localization of OS1-179(+) and OS1-179(−) ncRNAs (blue arrows), as compared to _Emx2OS_-ncRNA and _Emx2_-mRNA (for sake of clarity, the sense/antisense ovarlapping region and its surroundings are represented enlarged; red, non coding sequences; violet, coding sequences).