The overall goal of this procedure is to generate and select stable trenchgenic lines of the novel model organism, Austria Occus Tori, a marine pico eukaryotic lgal species. This is accomplished by first generating cell pellets from 50 milliliters of dense ALG culture per transformation. The second step is to mix the cells with linearized DNA and then to perform the electroporation.
After diluting the transform cells in fresh, medium and incubating overnight, add 0.2%low melting point aase and a selective agent, and pour the mixture into small Petri dishes. The final step is to pick resulting colonies that will have grown after two to three weeks incubation in constant blue light. Ultimately selection in liquid, medium, and subsequent molecular analyses such as PCR and southern blot are used to show successful integration of the construct into the genome of the generated transgenic lines.
The main advantage of using this organism over existing model organisms like Arabidopsis is to greatly reduce genomic complexity that allows the study of regulatory networks in the background with virtually no genetic redundancy. Although in my lab we've used osteo occus to study the plant circadian clock, it can be used to study many other complex cellular networks. So the implications of this technique extend well beyond the realm of plant biology.
Visual demonstration of this method is critical because although the individual steps appear rather simple, proper handling of the cells and an efficient workflow are essential for obtaining a good result. Parts of this procedure will be demonstrated by Culin Kish, a research assistant from a laboratory. Austria Occus cells are accultured in artificial seawater or a SW to prepare a SW dissolve 40 grams per liter.
Sea salts in deionized water to a salinity of 30 parts per thousand or PPT as measured. Using a salinity meter add enrichment, medium trace metal elements and vitamins, and then filter sterilized through 0.22 micron filter gross cells of Austria cocus strain O TT H 95 in a SW under constant light. In a plant growth incubator fitted with a moonlight blue filter, the light intensity should be close to 20 micromoles per meter square per second, and the temperature maintained at 20 degrees Celsius cells do not require constant agitation, but are shaken once every two to three days to prevent aggregation.
For maintenance, subculture cells aseptically at a dilution of one to 100 in fresh a SW every seven days. Each transformation requires 50 milliliters of cells at a cell density of 20 to 30 times 10 to the six cells per milliliter. This density should be attained five to seven days following subculture.
Approximate cell density and axe can be estimated on a hemo cytometer at a minimum of 40 times. Magnification or preferentially be determined by flow cytometry. An example of healthy versus unhealthy cultures is shown here.
Each bio parametric plot shows side scatter or SSC on the y axis versus red fluorescence per cell or FL three on the x axis. FL three represents chlorophyll red fluorescence, and SSC is indicative of relative cell size. Healthy cells fall into the Austria Occus window, whereas dying cells or contaminating bacteria fall outside this window.
Ideally, more than 99%of the cells should be in the Austria Occus window. For efficient genetic transformation, each transformation requires five micrograms of pure linearized plasmid, DNA as a concentration of one microgram per microliter. In sterile deionized water, the plasmid has been linearized by an enzyme that cuts in the backbone of the vector used, but not in the transgene or selection gene.
Keep the micro centrifuge tube containing DNA on ice together with a two millimeter electroporation vete for each transformation. A control with no DNA is necessary for each cell line to be transformed. For each transformation, transfer 50 milliliters of cells to a tube with a conical bottom add 0.1%onic acid F 68, and centrifuge for 10 minutes at 8, 000 times G at 10 degrees Celsius.
To pellet the cells immediately discard the sup natant and add one milliliter of Resus suspension buffer to the cells. Resus suspend the cells by pipetting up and down, transfer to a micro refuse tube and spin down for 10 minutes at 8, 000 times. G at 10 degrees Celsius working quickly wash the cells once more after the second spin.
Cut a P 200 tip to resuspend each pellet in 40 microliters of Resus suspension. Buffer at 40 microliters of the resuspended cells to each tube of linearized DNA on ice. Mix gently and transfer to the electroporation vete.
Put the vete in the electroporation machine. Adjust the settings to six kilovolts per reciprocal sensor, 600 ohms and 25 micro days. Electro operate the cells after electroporation.
Incubate the cells in the cuvettes at room temperature for 10 minutes. During this time, label the tissue culture flasks and add 30 milliliters of fresh a SW to each flask. Take one milliliter of a SW out of each flask and gently add it to the corresponding vete incubate for two minutes.
Next, gently remove the a SW that now contains the glo of cells and gently and slowly pet directly into the a SW.In the culture flask cells will typically remain in a big Glo. Take care not to shake or disturb the aggregated cells at this moment. Allow the cells to recover in the incubator for one to two hours.
Finally, resuspend the cells by manually shaking the flask. At this point, the cells should resuspend freely and no clumps should be visible. Leave the cultures to recover overnight in the incubator on the day following electroporation autoclave a solution of 2.1%low melting point haase in double distilled water in a bottle containing a star rod.
Keep molten at 65 to 90 degrees Celsius in a larger beaker containing water on a heating magnetic starr. For each transformation, prepare eight 50 millimeter Petri dishes and eight 50 milliliter tubes that each contain nine milliliters of a SW plus. The required selection.
Collect the transform cells from the incubator. The most difficult part of this procedure is the inclusion of transformed cells into low percentage agros gels without breaking the integrity of the cells by exposing them to too much heat. So it's important to perform the next few steps of the procedure quickly and efficiently Working in a sterile flow hood.
Add one milliliter of near boiling low melting point aase to the nine milliliters of a SW.In one of the tubes, close the tube and invert to mix. Next, add 0.5 milliliters of freshly transformed cells. Quickly close and invert the tube to mix and then pour its content into the Petri dish.
Repeat this process with the remaining seven tubes and then proceed to the next transformation flask. Leave the dishes open in the flow hood for an hour for the aray to set. Then cover the dishes and transfer them carefully to large square Petri dishes that each hold four round dishes.
Since the dishes were not set completely, the gel is very fragile. Be careful not to break the gel when handling the dishes. Seal the square dishes with paraform and place them in the incubator.
Once colonies appear, use a 200 microliter perpe with cutoff tips. To pick colonies in a sterile hood, simply select free colonies and suck out a green colony. Take care not to include any cells from neighboring colonies.
Transfer the cells to two milliliters of liquid medium containing the selection. In a 24 well plate mix by petting up and down. After selecting 24 or 48 colonies per transformation, seal the plates with para film and transfer to the incubator.
After a week transfer 100 microliters of each well to two milliliters of fresh a SW with selection. In a 24 well plate grow for an additional seven days. Surviving cell lines can then be used for further studies when transforming five micrograms of linearized DNA.
Using this protocol. 20 to 60 colonies per transformation plate should appear after two to three weeks. Approximately 80%of colonies picked were positively selected by antibiotic resistance in liquid medium, and were used in subsequent studies Following this procedure.
Other methods like PCR or Southern blot can subsequently be performed in order to verify insertion number and location of the trench gene After its development. This technique allowed researchers in the circadian clock and cell cycle fields to understand the complex components and behaviors of their respective systems In this new model organism, Austria Caucus. Tori, After watching this video, you should have a good understanding of how to generate transgenic Austria caucus lines.
