We demonstrate the successful expansion of the genetic code in a metazoan with components derived from two different E. coli
tRNA/RS pairs. Uaa incorporation is nonlethal to the transgenic worms and shows dosage, temporal, and temperature dependence. The mCherry fluorescence and luciferase activity we observed in C. elegans
were Uaa-dependent with dosage and on-and-off switch effects, indicating that reporter protein expression is dependent on the incorporation of the Uaa. The different tRNA/RS pairs, Uaas and target proteins we employed suggest that our approach can be generally applied to various Uaas and proteins. The tyrosyl and leucyl tRNA/RS pairs have been evolved to incorporate a large number of Uaas and widely used in E. coli
, yeast and mammalian cells (9
); our demonstration of their compatibility in C. elegans
should enable the genetic encoding of these reported Uaas in worms. Strikingly, important concerns when expressing Uaa-containing proteins in C. elegans
have been uncovered in this research, including the transgenesis techniques used to express the target protein and the methods used to deliver the Uaa to the cells of interest.
A common challenge for incorporating Uaas in eukaryotes is the expression of a functional orthogonal tRNA, which often lacks the intragenic promoter elements conserved in eukaryotic tRNAs necessary for transcription by Pol III. We previously developed a general method to express orthogonal tRNAs by using a post-transcriptionally cleavable Pol III promoter in yeast (3
) and type-3 Pol III promoters in mammalian and stem cells (5
). Here we demonstrate that this strategy can be extended to C. elegans
. The rpr-1
promoter efficiently drives the expression of E. coli
for functional translation in worms. The rpr-1
promoter is a homolog of the H1 promoter in human cells that we used to express orthogonal tRNAs in mammalian cells (5
). Both promoters are type-3 Pol III promoters for expressing the RNA component of RNaseP, which is involved in tRNA processing and required for all cells at various developmental stages. Therefore, use of the rpr-1
promoter for orthogonal tRNA expression should be generally compatible with Uaa incorporation in different tissues and stages in C. elegans
. Recently Greiss et al.
demonstrated the use of another Pol III promoter, the cen74-1
promoter, to express the M. mazei
in worms (18
There are several methods used to introduce transgenes in C. elegans
. We have discovered that a target gene containing a UAG codon in C. elegans
is most reliably expressed from a chromosomally integrated low copy number construct. Both microinjection and microparticle bombardment can result in the formation of extrachromosomal arrays containing multiple copies of the transgene for high-level expression. When a UAG-containing gene is expressed in an extrachromosomal array, background UAG readthrough is detectable in the absence of an amber suppressor tRNA. Low levels of stop codon readthrough have been reported in C. elegans
) and are confirmed by our observation of red fluorescence in worms injected with only the mCherry amber reporter. Nonspecific UAG readthrough is possibly due to insertions or deletions near the UAG and/or context codons, which can occur during the recombination that forms the extrachromosomal array (19
). In contrast, single-copy integration of the same mCherry reporter gene into a chromosome showed absolutely no background red fluorescence. Consistently, single integration of the luciferase amber reporter into a chromosome showed virtually no luciferase activity. Therefore, a chromosomally integrated, low copy, sequence confirmed UAG-containing reporter gene is important to maintain high fidelity when reporting amber suppression and Uaa incorporation.
A critical component of effective and experimentally relevant Uaa incorporation is the generation of animals with a high level of expression and a minimum of genetic mosaicism. The rate of generating transgenic worms in Greiss's report is very low (1–5 per hundreds of worms), and even within an established line maintained with antibiotics, only 5% of the animals containing the transgenes express the reporter. The low success rate in generating animals harboring introduced genes in combination with the high level of mosaicism due to array transmission would make it nearly impossible to identify a strain expressing an exogenous gene that does not have a sensitive and straightforward readout such as fluorescence. In contrast, our integrated single copy reporter had zero false-positive signals. When an orthogonal tRNA/RS was injected into the reporter line, we obtained strains that incorporate Uaas with a success rate of 25–50%. To achieve the genetic uniformity of our animals, we integrated all of the genetic components into chromosomes and bred the worms to homozygosity, enabling the transmission rate of our transgenes to be 100%. Each of these transgenic worms showed Uaa-dependent responses at reproducible levels in the tissue targeted. Our high transmission and incorporation rate should facilitate the generation of transgenic worms to incorporate Uaa into different target genes.
Low-copy integration of amber-containing target genes is desirable for the generation of Uaa-containing target proteins to investigate biological processes in C. elegans. Nonspecific amber readthrough in extrachromosomal arrays can result in natural amino acids at the amber site in the target protein, and this lack of fidelity when incorporating Uaas will impair rigorous study of the target protein. In addition, array expression results in inconsistency both between animals of the same genotype and within a single animal, which can make results difficult to interpret. By integrating the extrachromosomal array expressing the orthogonal tRNA/RS pair, we generated a population of genetically identical animals, and abrogated the need to constantly select for transgenic animals. A stable transgenic worm capable of Uaa incorporation should facilitate analysis wherein mosaic transgenic worms can impose complications. Moreover, the ability to detectably suppress the amber codon in a single-copy gene suggests a fairly high efficiency for our Uaa incorporation machinery. Therefore, we may be able to recapitulate natural levels of gene expression, rather than overexpression, when generating Uaa-containing proteins. This close-to-native condition will help obtain physiologically relevant results.
A multicellular organism such as C. elegans imposes more challenges on the bioavailability of Uaa than single cells. DanAla, a Uaa deviating from natural amino acids in structure, was sequestered in the intestine when unmodified. Supplying DanAla in dipeptide form enables DanAla to traverse the intestine and reach muscle cells, suggesting that dipeptide transporters tolerate structural deviations of substrates more than amino acid transporters. Because DanAla is so drastically different from natural amino acids, successful uptake in dipeptide form suggests that other Uaas can be delivered into C. elegans using this strategy. As dipeptide transporters are present on most of C. elegans cells, this method has the potential to deliver Uaas to different tissues.
The development of a multicellular animal involves many complex interactions between proteins. Genetically encoded Uaas will afford new chemistries to enable finer investigation and manipulation of protein functions directly in living C. elegans. Our strategies should be generally applicable for the incorporation of various Uaas in worms, and should be valuable in guiding the genetic code expansion of other multicellular organisms.