Expanding The Genetic Code Of Vertebrates With Unnatural Amino Acids

The vast majority of natural life forms on earth conservatively use the standard genetic code to specify 20 canonical amino acids.While minor code variations are naturally found in bacteria and eukaryotic cells,artificially expanding the genetic code to utilize unnatural amino acids(Uaas)in vertebrates is challenging.The genetic code can be expanded to encode unnatural amino acids(Uaas)by introducing an orthogonal suppressor tRNA/aminoacyl-tRNA synthetase(aaRS)pair to decode a blank codon(usually is stop codon or frameshift codon).Initially developed in Escherichia coli,this strategy has been proven generally applicable in eukaryotic cells and in generating transgenic invertebrates capable of Uaa incorporation,including Caenorhabditis elegans and Drosophila melanogaster.Although cells and tissues of mouse can be transduced transiently to incorporate Uaas,it remains unknown whether transgenic vertebrates with a heritable expanded genetic code system-wide can be generated,as it is unclear whether the biological complexity of vertebrates allows the introduction,maintenance and transmission of the newly introduced genetic material for code expansion.We firstly decided to integrate the orthogonal AzFRS/tRNACUA pair,which can decode the amber stop codon UAG as the Uaa p-azido-phenylalanine(AzF),into the mouse genome.The optimized RS(tRNA)4x construct was linearized and directly delivered into nuclei of mouse zygotes,we succeeded in constructing RS(tRNA)4x transgenetic mouse,and RS(tRNA)4x gene was transmitted efficiently to subsequent generations.On average 15 copies of transgenes was detected in an F2 mouse as determined by qPCR using GAPDH as the internal control,and the expression of AzFRS proteins was detected in various tissues in F2 mice by western blotting.These date indicating that the transgene was integrated into the mouse genome and stably transmitted.We next evaluated the potential impact of gene integration in the living mice.Through HE histology analysis,no detectable morphological differences were observed between transgenic mice and the littermate controls.We used RNA-seq to analyze the transcriptomes of the liver tissue,where AzFRS showed the highest expression.In comparison with the WT mice,we found minor change in gene expression.To test whether these small expression changes impaired the liver function in the transgenic line,we measured blood chemical indexes,No significant difference was observed between WT and transgenic mice.In addition,we fed the transgenic mice with or without AzF,and no obvious abnormalities in blood chemical indexes were observed.Taken together,these results demonstrate that genome integration of RS(tRNA)4x gene in mice did not cause significant physiological impairment.To functionally evaluate the integrated AzFRS/tRNAcuA pair,AzF incorporation experiments were performed in adult transgenic mice-derived primary cells with transduction of an EGFP-amb-mCherry reporter.After adding AzF into cell culture medium,amber suppression was detected in differentiated neurosphere and primary bone marrow cells from transgenic mice but surprisingly absent in fibroblasts.However,when we cotransfected additional tRNAcuA with the EGFP-amb-mCherry reporter into fibroblasts derived from transgenic mice,AzF incorporation was detected,suggesting that AzFRS was functional and tRNAcuA expression may be limited in fibroblasts.Taken together,these data demonstrate the successful expression of functional AzFRS/tRNAcuA in primary cells isolated from the transgenic mice,which directs AzF incorporation into the reporter in response to the amber stop codon.AzF incorporation was cell type-dependent,in part influenced by the amount of tRNAcuA expressed.We also sought to expand the genetic code in zebrafish.A construct for ubiquitous expression of AzFRS/tRNAcuA was co-injected with the mRNA of To12 transposase into zebrafsh embryos at 1-cell stage to generate the transgenic fish.AzFRS expression was detected via its C-terminal FLAG tag in both larvae and adult caudal fn in F2 generation,revealing the successful and stable integration of the transgene.To assess AzF incorporation in vivo.In vitro-transcribed mCherry-eGFP(Y145amb)mRNA was injected into F2 transgenic fish embryo at 1-cell stage,and the embryos were incubated in water containing 2.5 mM AzF.In the presence of AzF,there was a significant increase in the number of cells with eGFP signals 24h post ferlization(hpf),indicating suppression of the amber stop codonIn conclusion,we for the first time report the successful generation of transgenic mice and zebrafish with an expanded genetic code.The orthogonal synthetase and tRNACUA directed Uaa incorporation in response to the amber stop codon ex vivo in mice and in vivo in zebrafish.Transgenic vertebrates genetically exploiting Uaas will be invaluable for studying physiological and pathological processes as well as creating unique functions in vivo.