Worm ttTi5605 Locus Expression Vector

The introduction of a single copy of an exogenous gene at precise locus is a powerful tool that has been widely used in the model organism C. elegans. The ttTi5605 locus expression vector is a well-characterized and highly effective system for generating site-specific single-copy insertions of transgenes in C. elegans. This system can induce stable integration of transgenes using either Mos1 transposon excision or CRISPR techniques.

The Mos1 transposon was discovered in Drosophila mauritiana. A large cohort of C. elegans strains has been developed to contain the Mos1 transposon inserted at defined sites. One example of this is the ttTi5605 genome region of C. elegans which carries the Mos1 site (https://wormbase.org/species/c_elegans/variation/WBVar00254893#02-456-10). The ttTi5605 locus was chosen as the integration site because the insertion does not disrupt the function of neighboring genes. In addition, the Mos1 transposon excision technique results in insertion of a single copy of the user’s transgene of interest, which is not easily silenced by the RNAi system in the worm. For Mos1 induced ttTi5605 site specific gene recombination to occur, a helper plasmid coding for a transposase is co-transfected with the ttTi5605 locus expression vector. Transcription of the transposase results in excision of the Mos1 transposon from the worm genome. The double-strand breaks are then repaired with the external provided sequence, causing stable genetic recombination. With Mos1-mediated Single Copy Insertion (MosSCI), the gene of interest is integrated at the targeted ttTi5605 site on chromosome II in C. elegans.

The ttTi5605 locus expression vector includes many important components that enable efficient expression of your gene of interest. First, our promoter database helps you choose the best promoter for your application and includes ubiquitous, tissue-specific, and inducible heat shock protein (HSP) promoters. We offer spec-1, myo-2, and myo-3 which have been demonstrated to lead to tissue-specific gene expression in C. elegans. The second feature of our vector is that a 3’ UTR with a polyA signal is integrated to function post-transcriptionally to regulate protein expression. Finally, the wild-type C. Briggsae unc-119 gene is placed between the flanking DNA as a positive selection marker. Unc-119 (ed3) mutant worms are small, almost paralyzed, have small brood sizes and are incapable of forming dauer larvae when starved. Thus, after inducing transgene integration, the rescuing animals will be those carrying the extrachromosomal DNA.

Repair of the double-strand breaks generated by excision of a Mos1 transposon is limited by relative scarcity of Mos1 insertion sites in the genome. In contrast, a much wider range of genome modifications is made possible by using a CRISPR-based technique. Our ttTi5605 locus expression vector system can also be used for Cas9-triggered homologous recombination. For this application, plasmids that encode a sgRNA targeting a sequence adjacent to the ttTi5605 site and Cas9 protein should be transfected into worms.

For further information about this vector system, please refer to the papers below.

Permanent integration of vector DNA: Transfection of worms with regular plasmid gene expression vector results in transient delivery of DNA into host cells due to the loss of DNA overtime. In contrast, transfection of cells with the ttTi5605 locus expression vector can induce stable transgenes due to the integration of the transgene into the host genome. This enables precise sequence editing and helps construct genetically modified worms.

Technical simplicity: Delivering plasmid vectors into worms is technically straightforward and far easier than virus-based vectors which require the packaging of live virus. Either electroporation-based delivery or microinjection of plasmids into germ cells or simply feed worms with bacteria transformed with the plasmids results in transgene expression.

ttTi5605_LA: Left homology arm (1,336 bp) targeting C. elegans ttTi5605 locus.

Promoter: The promoter driving your gene of interest is placed here.

Kozak: Kozak consensus sequence. This is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

3' UTR+polyA: Allows transcription termination and polyadenylation of mRNA transcribed by RNA polymerase II. It functions in somatic and germ cells.

Unc-119(+): Facilitates the use of the unc-119 mutant rescue as a selection marker for transgene insertions when using C. elegans unc-119 mutants as the background.

ttTi5605_RA: Right homology arm (1,428 bp) targeting C. elegans ttTi5605 locus.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin:  Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.