2A ribosome skip sequence selection

<div>To verify the ribosome skip capability of the "2A" sequences in mammalian cells, several constructs were cloned: constructs with the T2A sequence and various linkers downstream of it as well as a construct with the P2A sequence. The constructs were sequence verified prior to experimentation. When they were deposited at Addgene, they were also NGS sequenced in their entirety for their QC process. All plasmids passed the QC. The plasmids are available at http://www.addgene.org/browse/article/28196220/. The individual NGS sequences can be obtained by clicking each individual construct, for example, http://www.addgene.org/113839/sequences/. The constructs were then tested by calcium phosphate transfection of 293T transformed human embryonic kidney cells.</div><div><br></div><div>In the constructs, a nuclear localized Histone H2B-sfGFP and a tdTomato without any localization signal were fused by the 2A sequence to visually confirm the 2A skip. The idea is that if the 2A skip is efficient, the second protein would fill the cell diffusely without a significant enrichment in the nucleus. If the 2A skip is inefficient, the second protein would be enriched at the nucleus along with the first protein. This is similar to experiments in Ahier and Jarriault, 2014 (https://doi.org/10.1534/genetics.113.160846), although I wasn't aware of this paper at the time of my experimentation (January of 2016).<br></div><div><div><br></div>As one can see in the attached pdf files, all T2A constructs resulted in nuclear sfGFP fluorescence and diffuse tdTomato fluorescence. In contrast, the P2A construct resulted in nuclear sfGFP fluorescence and nucleus-enriched tdTomato fluorescence. The relative efficiencies of the 2A sequences are debated. As shown above, in my hands, the T2A seemed to outperform the P2A in this assay in human and mouse cells. Further characterization of the T2A-mediated ribosome skip utilizing a FRET analysis (Document attached to Addgene plasmids.pdf) showed that the T2A skip was indeed efficient. Thus, I chose the T2A for my mouse lines. In the end, the T2A "worked" just fine, as is evident from images of the mouse lines in the data collection here. <br></div><div><br></div><div>I am not sure what caused my observations with the P2A. I also tried the "PQR" variant 3 P2A (https://doi.org/10.1016/j.celrep.2015.11.048). It behaved the same as my P2A sequence in this assay. Perhaps there are other sequences that are necessary for or inhibitory to the P2A function. Regardless of these findings, a mouse line with a P2A-creER^T2 obtained from Jax "worked" just fine too (not shown). In the end, I am not sure what is going on with my P2A, but I am not pursuing the P2A further since my T2A seems to work just fine as is in my hands.<br></div><div><div><br></div><div>Interestingly, Suzuki et al., 2019 (https://doi.org/10.1538/expanim.19-0031) seems to show nuclear localization of the fluorescent proteins after the P2A (i.e., co-localization of the fluorescent protein signal with presumably nuclear transcription factor signal). Another paper Yoshimatsu et al., 2021 (https://doi.org/10.1016/j.scr.2021.102308) also seems to show nuclear enriched fluorescent protein after the P2A. But it's difficult to interpret those images.</div><div><br></div></div><div>The tiny particles are calcium phosphate:DNA precipitate, not bacterial contamination.<br></div>