| Strongyloides stercoralis is a common,although often focally distributed,intestinal nematode parasitizing dogs,human,and other primates.In contrast to the majority of other animal parasitic nematodes whose adult stages are confined to the definitive host,Strongyloides spp.and related genera have one or more generations of free-living males and females that exchange genetic material during sexual crosses.Transfer of plasmid-encoded transgenes by microinjection into the syncytial gonads of female hermaphrodites became the primary means of gene transfer in the model nematode Caenorhabditis elegans when this method was pioneered in the 1980 s and it has remained so to date.The body plans of free-living strongyloidoid females are strikingly similar to those of C.elegans hermaphrodites with amphidelphic ovaries that have syncytial zones in their distal ends,and this has permitted adaptation of microinjection techniques for gene transfer in C.elegans to strongyloidoid parasites with few modifications.There are currently no effective vaccines available for these parasites,and chemotherapy is based on a relatively small arsenal of anthelmintic drugs.Resistance to each of these is widespread among parasites of livestock,and suboptimal anthelmintic treatment responses in human clinical settings may signal genetically based resistance arising in populations of nematode parasites of humans.Screening has and will likely continue to identify new candidate anthelmintics against nematodes and introduce new vaccine.The most widely used version of this approach,hereafter referred to as the CRISPR/Cas9 system,involves mutagenesis catalysed by a complex of the Streptococcus pyogenes CRISPR-associated(Cas)endonuclease Cas9 and a small guide RNA(sg RNA)comprising trans-activating and sequence-specific targeting domains.Deploying RNA-guided nucleases based on CRISPR to introduce insertions or deletions(indels)in target genomic sites represents the state of the art in genome editing and gene disruption in eukaryotic cells and whole organisms.The CRISPR/Cas9 system has been adapted for use in non-parasitic nematodes C.elegans,the protozoa and the necromenic nematode Pristionchus pacificus.I have recently achieved proof of principle for targeted mutagenesis in S.stercoralis via CRISPR/Cas9.As animal host is required for the establishment of the stable transgenic lines of S.stercoralis,I also have optimized the system in S.stercoralis – infected Mongolian gerbil and for the first time established the S.stercorlias infection in Meriones meridianus.(1)Optimization of animal model scheme of S.stercoralis-infected Mongolian gerbil,establishment of the animal model scheme of S.stercoralis-infected Meriones meridianus for the first time.My research work has optimized animal model scheme of S.stercoralis-infected Mongolian gerbil,including reducing the number of i L3 for infection(from 1000 to 170),decreasing the frame time of recovering larvae from feces(from 35 days to 11 days),improving the efficiency of infection(from 6.5% to 77.8%).These work made the system more suitable for transgenic research.Also I established the animal model scheme of S.stercoralis-infected Meriones meridianus for the first time,including DAPI staining of S.stercoralis parasitic female(PF),morphological observation of S.stercoralis PF,L3 a and parasitic fourth-stage larvae(PL4),tested the blood physiological indexes and blood biochemical indexes,hematoxylin-eosin staining of intestine of S.stercoralis-infected M.meridianus,M.gerbil and Begal.(2)Genetic transformation of S.stercoralis by microinjection of plasmid DNA constructs into the male germlineThe promoter of Ss-rps-21 was constructed into the plasmid which included GFP+Ss-era-1.The plasmid was named p AJ20,which were then microinjected into the free-living female gonad and male germline,respectively.The anatomic expression patterns showed ubiquitous distribution of GFP in all body tissues with the highest level of expression occurring in the genital primordium(GP),but the highest level of expression occurring in the head and tail of transgenic F1 by transforming male germline.The position for injection in males and the plasmid concentrations were determined at pharynx and 900-1000 ng/μl.(3)Gene edition via CRISPR/Cas9 system in S.stercoralisThe codon usage from S.stercoralis was calculated and optimized for Ce Cas9 protein based on the preference of codons in order to express Cas9 protein in S.stercoralis.In addition,15 U6 genes were retrieved from S.ratti genome sequence in Wormbase and multiple protein sequence alignment of Sr-U6 to find conserved domain and select other conserved sequence from upstream and downstream regions.The g RNAs were designed to target the exon1 in Ss-dpy-2.To verify the efficiency of g RNA,the enzyme digestion assay was performed.The results demonstrated that the gRNA4,7 and8 were more efficient.To verify expression of Ss Cas9 and g RNA in S.stercoralis,the gonads of wild-type free-living adult worms were microinjected with these plasmids,generating transgenic progeny that carried each expression vector alone or both in stableextrachromosomal arrays.Total RNA was extracted from these transgenic lines and reverse transcription(RT)-PCR assays was conducted to detect the transcripts.These results confirmed that Ss Cas9 and g RNAs were transcribed in transgenic worms,indicating that the Ss-rps-21 and Sr-U6 promoters in the plasmid vectors were active.Furthermore,3 homologous repair templates aiming at gRNA4,7and 8 were constructed.p AJ50-Cas9 and p XL-BACII-g RNA with corresponding homologous repair template were then microinjected into female gonad,then the target position was amplified by Nested-PCR which produced no band.Amplification of the exon1 of Ss-dpy-2 and sequencing,there is no impure peaks,indicating that the gene of Ss-dpy-2 does not occur mutation.In conclusion,I have optimized animal model scheme of S.stercoralis-infected Mongolian gerbil,established the animal model scheme of S.stercoralis-infected Meriones meridianus for the first time,achieved genetic transformation of S.stercoralis by microinjection of plasmid DNA constructs into the male germline and obtained the anatomic expression pattern of Ss-rps-21 in transgenic F1 by transforming the male germline.I have also attempted establishing gene edition via CRISPR/Cas9 system in S.stercoralis,verified that Ss-rps-21 and Sr-U6 promoters in plasmid vectors were active.The achievement from this thesis provides a good basis for subsequent research in transgenesis of parasitic nematodes and other parasitic helminths. |
