Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

The overall goal of this procedure is to rescue infectious virus particles made entirely from a cloned CDNA of positive strand, RNA viruses whose genomes are in the same polarity as cellular Mr.NA, such as the Japanese encephalitis virus. This CNA based regene genetics is a semial method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic study in viral replication and pathogenesis. This technique also provide a valuable platform that allows the development of genetically defined vaccine and viral vectors for the delivery of foreign genes.

These vessels are applicable to cloning full length CDNA for length or positive strain, RNA viruses, particularly those with genomes larger than 10 kb. Demonstrating the procedure will be several members of my laboratory research assistant Professor San Nun to postdoctoral fellow UNG and Xing y Kim and the undergraduate students, Jordan Frank. The video portion of this publication covers the preparation of the bacterial artificial chromosome containing the sequences of interest, the synthesis of RNA, transcribed from the back plasmid, and how to measure the RNAs infectivity, and the virus yield.

All the procedures used prior to generating the back plasmid are provided in the text protocol in great detail. For this procedure, first grow a single colony of e coli DH 10 B, carrying the back plasmid in three milliliters of two XYT broth with chloramphenicol. Incubate the culture for 10 hours at 35 degrees Celsius with shaking at 225 to 250 RPM.

After 10 hours, scale up the culture, then inoculate 500 microliters of growth into 500 milliliters of medium and cultivate the inoculum for six hours with vigorous shaking after six hours. Centrifuge the bacterial culture in 2 250 milliliter bottles at 3, 100 Gs for 15 minutes at four degrees Celsius. Then resuspend each pellet in 30 milliliters of GTE solution.

Add 500 microliters of lysozyme and incubate the suspensions on ice for 10 minutes. In the meantime, prepare fresh lysis solution for the next step. After 10 minutes, add 60 milliliters of the lysis solution and mix by swirling until the solution appears clear.

Let the reactions go at room temperature for 10 minutes. Next, add 45 milliliters of neutralization solution to each bottle and invert the bottles until they are thoroughly mixed. Then put them on ice for 10 minutes.

Collect the neutralized lysates with the high G refrigerated centrifugation. Then collect the supernatants to two new 250 milliliter bottles. Add 0.6 volumes of 100%isopropanol to each and put them on ice for 20 minutes.

Next, spin down the precipitates, then discard the supernatants and dissolve the pellets in five milliliters of TE buffer. Combine the two volumes in a single 50 milliliter tube and precipitate the RNA by adding an equal volume of five molar lithium chloride and incubating the mixture on ice for 10 minutes. Use a 20 minute cold centrifugation to collect the RNA precipitate.

Then transfer the supernatant to a new 250 milliliter tube. Add two volumes of 100%isopropanol and put the tube on ice so the DNA precipitate will form. After 20 minutes, spin down the precipitate, aspirate the supernatant, resuspend the DNA pellet in 9.5 milliliters of TE buffer and transfer the DNA solution to a 50 milliliter tube.

Continue the preparation by setting up the cesium chloride gradient with a th dium bromide. Load the mixture into a sealable polypropylene tube using a syringe with an 18 gauge needle and seal the tube. Spin the sealed tube overnight in an ultracentrifuge to form a cesium chloride gradient.

The next day collect a DNA band of back plasmid from the cesium chloride gradient. Use an 18 gauge needle to create an air vent at the top of the gradient and a 20 gauge needle with syringe to collect the back DNA from the side of the gradient, and then transfer the collection to a 1.7 milliliter micro fuge tube. Perform a wash, add 2.5 volumes of water saturated butanol and mix with normal vortexing.

Then centrifuge the mixture at room temperature and transfer the lower aqueous phase to a new 1.7 milliliter micro fuge tube. Repeat this wash a total of six times to remove all the ethidium bromide. Now precipitate the back DNA.

Add a one 10th volume of three molar sodium acetate and 2.5 volumes of 100%ethanol to the tube. Incubate the mixture on ice for 10 minutes. Then centrifuge the precipitate at room temperature and wash the DNA pellet with one milliliter of 70%ethanol.

Repeat the spin and collect the pellet again. Finally, air dry the pelleted DNA for 10 minutes and ute it in 200 microliters of TE buffer. Begin with a large scale restriction enzyme digestion of the back plasmid with xbo.

One in a total volume of 100 microliters. Perform this digestion at 37 degrees Celsius for 12 to 15 hours. Next, further incubate the digestion with an additional 25 units of mung bean nuclease at 30 degrees Celsius for two hours.

After the incubation, bring the volume up to 300 microliters with distilled water. Now perform a phenol extraction by adding an equal volume of phenol chloroform, isoamyl alcohol to the diluted sample. Vortexing vigorously for a minute, spinning down the tube and transferring the upper aqueous phase to a new tube.

Then repeat the extraction with chloroform. Next, recover the linearized back DNA by ethanol precipitation. Collect the DNA pellet with centrifugation, discard the supernatant and wash the pellet with one milliliter of 70%ethanol.

Repeat the centrifugation step and air dry the pellet. After 10 minutes of drying, dissolve the DNA pellet in 30 microliters of distilled water. Then examine one microliter of the recovered back on a 0.8%AROS gel.

Next, set up a 25 microliter runoff transcription of the linearized back DNA, including tritiated UTP for RNA quantification. Perform the reaction at 37 degrees Celsius for an hour. Then examine one or two microliters of the runoff transcription reaction on a 0.6%AROS gel.

Start with BHK 21 cells grown for 24 hours in 150 millimeter dishes plated at 3 million cells per dish. Rinse the cell monolayer with cold solution A and then detach them by trypsin.Ization. Collect the cells by centrifugation at 270 GS for two minutes.

Then resuspend the cells in 50 milliliters of cold solution A and spin them down again. Repeat this wash three times after the last wash. Resuspend the cells at 20 million per milliliter in solution A.Then mix a 400 microliter aliquot of the cell suspension with two micrograms of synthetic RNA in a two millimeter vete.

Promptly electro purate the mixture under optimal conditions and let the cells rest for 10 minutes. Then transfer the cells to a micro fuge tube containing 600 microliters of complete culture medium. Now prepare tenfold serial dilution of the electroporated cells in one milliliter volumes plate 100 microliter aliquots of each dilution on monolayers of normal bhk 21 cells after four to six hours of incubation, overlay the cells with 0.5%aros in MEM plus FBS and culture them for four more days.

Bhk 21 cells were mock electroporated or electroporated with RNA transcripts derived from two independent clones of the full length JEV Back four days later, the cells were stained with crystal violet to view the infectious RNA in the cells. Using this stain, the infectious centers were quantified. Next viral protein expression was examined in RNA Electroporated cells 20 hours post transfection using anti NS, one rabbit antis serum and a SI three conjugated secondary antibody seen in red.

The nuclei were counterstain with DAPI seen in blue. Later at 22 and 40 hours post transfection, the production of infectious varians accumulated in the culture. Supernatants of RNA electroporated cells was examined by plaque assays.

After watching this video, you should have a good understanding of how to rescue infectious virus entirely from a cloned CNA of of positive strain, RNA, such as Japanese encephalitis virus. So don't forget that working with a Japanese encephalitis virus and other human pathogens can be extremely dangerous. Proper biosafety training is necessary before performing this procedure.