Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection

Background Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines. Findings Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells. Conclusions The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin

Peptide and nucleic acid-directed self-assembly of cationic nanovehicles through giant unilamellar vesicle modification: targetable nanocomplexes for in vivo nucleic acid delivery

One of the greatest challenges for the development of genetic therapies is the efficient targeted delivery of therapeutic nucleic acids. Towards this goal, we have introduced a new engineering initiative in self-assembly of biologically safe and stable nanovesicle complexes (∼90-140 nm) derived from giant unilamellar vesicle (GUV) precursors and comprising plasmid DNA or siRNA and targeting peptide ligands. The biological performance of the engineered nanovesicle complexes were studied both in vitro and in vivo and compared with cationic liposome-based lipopolyplexes. Compared with cationic lipopolyplexes, nanovesicle complexes did not show advantages in transfection and cell uptake. However, nanovesicle complexes neither displayed significant cytotoxicity nor activated the complement system, which are advantageous for intravenous injection and tumour therapy. On intravenous administration into a neuroblastoma xenograft mouse model, nanovesicle complexes were found to distribute throughout the tumour interstitium, thus providing an alternative safer approach for future development of tumour-specific therapeutic nucleic acid interventions. On oropharyngeal instillation, nanovesicle complexes displayed better transfection efficiency than cationic lipopolyplexes. The technological advantages of nanovesicle complexes, originating from GUVs, over traditional cationic liposome-based lipopolyplexes are discussed. STATEMENT OF SIGNIFICANCE: The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. Giant unilamellar lipid vesicles (GUVs) have been used mainly as cell and tissue mimics and are instrumental in studying lipid bilayers and interactions. Here, the GUVs have been modified into smaller nanovesicles. We have then developed novel nanovesicle complexes comprising self-assembling mixtures of the nanovesicles, plasmid DNA or siRNA, and targeting peptide ligands. Their biophysical properties were studied and their transfection efficiency was investigated. They transfected cells efficiently without any associated cytotoxicity and with targeting specificity, and in vivo they resulted in very high and tumour-specific uptake and in addition, efficiently transfected the lung. The peptide-targeted nanovesicle complexes allow for the specific targeted enhancement of nucleic acid delivery with improved biosafety over liposomal formulations and represent a promising tool to improve our arsenal of safe, non-viral vectors to deliver therapeutic cargos in a variety of disorders

Urogenital Schistosomiasis and Sexually Transmitted Coinfections among Pregnant Women in a Schistosome-Endemic Region of the Democratic Republic of Congo

Schistosomiasis afflicts an estimated 10 million pregnant women in Africa annually. With mounting evidence of adverse impacts to reproductive health resulting from urogenital schistosomiasis, including increased transmission of HIV, further research on prenatal disease epidemiology is warranted, with implications for maternal and fetal health. Between October 2016 and March 2017, we conducted a cross-sectional study examining the prevalence of urogenital schistosomiasis and its association with sexually transmitted infections (STIs) other than HIV among pregnant women visiting antenatal clinics in Kisantu health zone, Democratic Republic of Congo. An extensive sociodemographic and clinical survey was administered to consenting participants, with urine samples and vaginal swabs collected to deduce active schistosomiasis and STIs, respectively. In total, 17.4% of expectant mothers were infected with Schistosoma haematobium, 3.1% with Chlamydia trachomatis (CT), 1.4% with Neisseria gonorrhoeae (NG), and 14.6% with Trichomonas vaginalis (TV). Women infected with urogenital schistosomiasis were at significantly increased odds of harboring a CT, NG, or TV infection (adjusted odds ratio = 3.0, 95% CI: 1.5, 6.0), but reports of clinical symptoms were low, ranging from 17.2% of schistosomiasis to 30.8% of TV cases. Laboratory confirmation of schistosomiasis and STIs provided objective evidence of disease in a cohort with low symptomology where syndromic management may not suffice. Shedding light on local risk factors and associated coinfections of urogenital schistosomiasis can identify unique intervention opportunities for prenatal care in trematode-endemic regions and aid in reducing adverse pregnancy outcomes