The mechanism of Ube3a imprinting: Relevance of sense - antisense transcriptional overlap between Ube3a and Ube3a-ATS
Zusammenfassung der Projektergebnisse
The aim of the project was to insert a transcriptional stop cassette into the "Ube3a" gene of the mouse to truncate transcription of the long non-coding antisense RNA "Snhg14" (former name "Ube3a-ATS"). Overlapping sense-antisense transcription of "Ube3a" and "Snhg14" is thought to be responsible for silencing of the "Ube3a" gene on the paternally inherited allele in neurons. This results in maternal-only expression of "Ube3a" in the brain. We decided to insert the transcriptional stop cassette in intron 1 of the "Ube3a" gene, to prevent transcriptional overlap at the promoter, in murine embryonic stem cells (mESCs). Because imprinted expression of "Ube3a" is observed only in neurons of the brain, the project also included the establishment of neuronal differentiation of mESCs. In order to follow "Ube3a" expression, we obtained mESCs carrying a gene trap lacZ cassette in the "Ube3a" gene from the German Gene Trap Consortium. We ordered two clones, E141C04 and P034A10. E141C04 has the lacZ gene trap inserted in intron 4, P034A10 in intron 1. Upon culturing and staining for lacZ expression we observed that the clones are not pure but a mixture with mESCs not carrying the lacZ trap. The lacZ trap is combined with a neomycin resistance gene, so we applied G418 selection for one week and isolated single clones, staining all blue. Neuronal differentiation of gene trap mESCs was achieved by culturing in neuronal differentiation medium containing N2/B27 supplement and EGF2 at later stages. Despite several attempts, cloning of the targeting construct was not successful, so that subsequent cell targeting experiments could not be performed. We cloned a 6kb homology fragment of "Ube3a" intron 1 into pBluescript, but subsequent integration of the combined polyA-stop-selection cassette into the homology fragment failed. Difficulties in cloning mainly included lack of bacterial colonies after transformation of ligations or failure to obtain the correct ligation product after plasmid preparation. Lack of bacterial colonies was due to problems with fragment isolation from agarose gels. We tested several different protocols for gel elution of DNA and obtained best results after freezing the gel slice before elution. The failure to obtain the correct ligation product might be inherent to the sequence of "Ube3a" intron 1. The patterns observed after restriction digests were reproducible but not predictable. We assume that recombination occurred between the vector backbone and the fragment used for ligation. Although our attempt to truncate "Snhg14" in mESCs was unsuccessful, we still believe that it is worthwhile to truncate the non-coding RNA before it traverses the "Ube3a" promoter. However, we would switch to the use of human induced pluripotent stem cells (iPSCs) and using the CRISPR/Cas9 system for inserting the stop cassette into "UBE3A" intron 1.
