Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Transient transaction of the strobilated forms of Echinococcus granulosus. We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors. Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines.

There is an urgent need for applied genetic research to understand mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of functional genetics of cestode parasites, including Echinococcus species. Advances in gene transferring in parasitic flatworms are still limited in comparison with protozoan parasite.

The use of viral delivery system has emerged as an essential tools for transgene delivery and gene/protein investigation over the last two decades. So we evaluated the lentiviral transient transduction of GFP reporter gene to the protoscoleces and strobilated worms of Echinococcus granulosus. Figure 1 demonstrates a schematic presentation of the study protocol for Echinococcus granulosus stages.

One, collecting hydatid cysts. Collect liver hydatid cysts from naturally infected sheep routinely slaughtered at an abattoir. Completely sterilize the cyst surface using sterile gauze and 70%alcohol before aspirating the protoscoleces.

Aspirate 20 to 50 mL of hydatid fluid out into sterile 50 mL conical tubes. After draining, blot out the cyst with a sharp blade, transfer the germinal layer into a sterile 50 mL conical tube, and shake it for a short period to increase the collection of protoscoleces. Wash the protoscoleces with PBS buffer three to five times.

Observe the protoscoleces in 0.1%eosin for five minutes to determine the protoscoleces'viability under a light microscope. Use protoscoleces with at least 95%viability for culture. Treat the protoscoleces precipitate with two mL of pepsin for 15 to 20 minutes.

To isolate individual protoscoleces, resuspend the protoscoleces sediment in PBS and pass it through two layers of sterile gauze into a new sterile 50 mL conical tube. Two, biphasic cultivation of protoscoleces of Echinococcus granulosus to obtain adult worms. Add 10 mL of the liquid phase medium to each 25 centimeter squared culture flask.

Add approximately 5, 000 protoscoleces to the culture flask and transfer it to the CO2 incubator. After 24 to 48 hours, transfer the protoscoleces to a new flask with the solid phase and add one mL of the same fresh liquid phase medium per flask. Transfer the flasks into the incubator, 37 degrees Celsius, 5%CO2.

Every five to seven days, replace the medium with fresh liquid phase medium. Three, monophasic cultivation of protoscoleces of a Echinococcus granulosus. Ensure that monophasic cultivation is done 24 to 48 hours before transduction.

Culture approximately 5, 000 protoscoleces in a 25 centimeter squared culture flask with RPMI and 10%FBS. Transfer the flasks into the CO2 incubator until use. Four, cell culture for virus production and preparation.

Transfer 500, 000 HEK cells to a 25 centimeter squared flask containing 5 mL of DMEM with 10%FBS, 100 U/mL penicillin, and 100 micrograms/mL streptomycin. Incubate the HEK2 cells at 37 degrees Celsius in 5%CO2 and passage them in the complete culture medium every 48 hours. Finally use low-passage HEK cells for transduction.

Five, production and preparation of the virus with the third-generation lentiviral vectors. Day one, after trypsinizing the HEK cells in a 6-well culture plates, seed 70, 000 cells per well with fresh antibiotic-free DMEM containing 10%FBS. Use cells at 50 to 60%confluency for transduction by the calcium phosphate method.

Day two, replace the cell culture medium two to three hours before the experiment with fresh antibiotic-free DMEM containing 2%FBS. In a 1.5 mL tube, mix third-generation lentiviral plasmids, including 7 micrograms/microliter pCDH513b transfer vector, 4 micrograms/microliter PLPII, 4 micrograms/microliter PLPI, and 2 micrograms/microliter PMD2G helper vectors. Add HEPES buffer to the mixture to reach the volume of 422 microliters.

Add 16 microliters of 1%TE buffer to the mixture. Add 62 microliters of 2.5 mL calcium chloride to the tubes and mix well. Add 500 microliters of 2x HBS buffer to the mixture, drop by drop within one to two minutes using a Pasteur pipette.

Mix gently until a semi-opaque solution is obtained and incubate the mixture for 20 minutes at room temperature. At the end, add the mixture to each plate slowly and distribute it with slow circular motions. Incubate the plates at 37 degrees Celsius in a 5%CO2 incubator and replace the medium after four to six hours with antibiotic free DMEM containing 10%FBS.

Day three, confirm successful transient transduction and cell viability using an inverted fluorescence microscope. Day four, harvest viruses from the culture medium by collecting each well's supernatant and store them at negative 70 degrees Celsius until use. Six, transient transduction of different stages of Echinococcus granulosus with the virus.

Day one, use a 12-well culture plate for gene transfer. Culture 5, 000 to 50, 000 HEK cells in triplicates with a complete DMEM medium as the internal reference. Culture 150 fresh protoscoleces in triplicates in RPMI and 10%FBS.

Culture 30 strobilated worms in triplicate into DMEM and 10%heat-inactivated FBS. Prepare a mixture of 1 million viruses with four micrograms/mL transfection reagent to transduce the HEK cells and different stages of the worm. Add the mixture to each plate slowly and distribute it with slow circular motions.

Incubate the plates for four to six hours in a CO2 incubator, 37 degrees Celsius, 5%CO2. Replace the medium for each sample with the same complete culture medium to minimize the toxic effects of the transfection reagents. Day two, repeat two previous steps to increase the efficiency of transient transduction for the HEK cells, protoscoleces, and strobilated worms.

Incubate all the plates for 24 to 48 hours in a CO2 incubator with the same culture conditions based on the type of cell media. Day three, make sure the transduction is successful by using fluorescence microscopy. Representative results.

Here we describe a rapid and efficient transient transduction technique in Echinococcus granulosus by using third-generation lentiviral vectors. We cultured protoscoleces in biphasic culture media to obtain strobilated worms according to our previous studies. Protoscoleces are developed into the strobilated worms after a six-week in vitro culture.

Different stages of Echinococcus granulosus were observed in the biphasic culture medium, including invaginated protoscoleces, Figure 2A, evaginated protoscoleces, Figure 2B, and strobilated worms with first and third proglottid formation, Figure 2C through E.Protoscoleces and the strobilated worms obtained from monophasic and biphasic cultivations, respectively, were transfected by GFP-expressing lentiviruses, Figure 3. HEK cells clearly expressed GFP as transient transduction process control. The adult worms expressed GFP most distinctly in the tegumental layer.

However, protoscoleces have demonstrated a somewhat lower level of GFP expression after 48 hours. Some cyst fluid residues and or germinal layer debris around the protoscoleces caused some fluorescence background in the transfected samples. The intensity of fluorescence in HEK cells and strobilated worms increased after 24 and 48 hours.

Understanding the molecular basis of nematodes and Platyhelminthes biology is one of the most important frontiers in the pathogenicity of zoonotic parasites. The lack of an effective gene evaluation system is a major obstacle to direct interpretation of functional genetics of cestode parasites, including Echinococcus species. The major goal of this work was to illustrate the process of lentiviral vector transduction which will be critical in future Echinococcosis research.

The results show that strobilated worms are more responsive to transient transduction of the gene than the protoscoleces, which may be the result of extensive tegumental structure in the strobilated forms. However, due to the nature of lentiviral transduction, the fluorescence intensity in the multicellular protoscoleces and strobilated worms is typically much lower than monolayer HEK cells. There is an urgent need for applied genetic research at the genomic and transcriptomic levels for Echinococcus as well as a wide variety of parasitic and free-living flatworm model systems.

Therefore, developing the gene transfer process to tapeworms paves the way for potential use of the new techniques such as virus-based gene mapping or CRISPR-Cas9-mediated gene editing. This work present lentiviral transduction in a tapeworm model and demonstrate promising outcomes with potential implication for flatworm biology. Yet more in-depth studies are required to improve the methods and develop the applicability of these techniques for future experiments.