五个抗生素抗性(AR)基因已被用于选择转基因真核细胞系,与BleoR,PuroR,HygR,NeoR,和BsdR盒赋予zeocin抗性,嘌呤霉素,潮霉素,遗传霉素/G418和杀稻瘟素,分别。我们最近证明,每个AR基因都建立了一个独特的转基因表达阈值,低于该阈值,没有细胞可以存活。BleoR选择最高水平的转基因表达,比使用NeoR或BsdR标记选择的细胞高出近10倍。这里,我们检验了AR蛋白功能与连锁表达之间可能存在成反比的假设,转基因编码,重组蛋白。具体来说,我们将每个AR蛋白融合到蛋白酶体靶向的Degron标签上,用这些来选择抗生素抗性细胞系,然后测量链接的表达,重组蛋白,mCherry,作为转基因表达的代理标记。在每种情况下,选择degron标记的AR蛋白的mCherry表达高于其同源WTAR蛋白。ER50BleoR入选最高水平的mCherry表达,比BleoR或任何其他AR基因高两倍以上。有趣的是,使用ER50BleoR作为选择标记翻译为更高,外泌体货物蛋白的外泌体负载增加3.5倍,CD63/Y235A。虽然是一种推定的CD63结合肽,CP05,已被用于装饰外泌体膜的技术被称为“外泌体绘画”,“我们在这里显示CP05与CD63-/-细胞的结合同样好,WT293F细胞,和过表达CD63的细胞,表明CP05可能非特异性结合膜。这些结果对于细胞工程特别是外泌体工程具有很高的意义。
Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/
G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as \"exosome painting,\" we show here that CP05 binds equally well to CD63-/- cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.