The overall goal of this protocol is to visualize step-wise procedures involved in the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with chemical and physical agents of interest. This test method can help answer key questions in the field of mutation research such as, is a test compound mutagenic? And if so, how potent of a mutagen is it?
Does it induce sequence-specific mutations? The main advantage of this technique is that it is a minimal to virtually any test compound. The method can be used for mutagenicity testing in mammalian cells using a chromosomally integrated reporter gene.
To constitute a single packaging reaction, add five micrograms of genomic DNA in a micro-centrifuge tube containing the first reaction mix. Then incubate the tube at 30 degrees Celsius for 90 minutes. Post-incubation, add 12 micro-liters of the second reaction mix to the tube and leave for another 90 minutes at 30 degrees Celsius.
After 90 minutes, add 1.1 milliliters of SM buffer to the tube. Then vigorously vortex the tube with the packaged DNA at room temperature for about 10 seconds. Quickly give a pulse spin in the micro-centrifuge.
Then leave the tube on ice until use. Next, add 50 microliters of chloroform for each milliliter of packaged DNA. If the sample will not be processed the same day, then vortex the sample gently and store at four degrees Celsius up to two weeks.
Prepare 16 sterile round bottom tubes and 16 TB1 agar plates for a single packaged DNA sample. Use ten tubes for screening and six for titering. Then aliquot 300 microliters of G1250 plating culture in each round bottom tube.
Next, to titer, add 10 microliters of packaged DNA to 990 microliters of SM buffer and 1:100 dilution. Then vortex the sample. Add 20 or 100 microliters of the 1:100 DNA solution to each of the three titer 20 or titer 100 tubes respectively.
For screening purpose, add 100 microliters of the undiluted packaged DNA sample to each of the 10 screening tubes. Process all of the 16 titering and screening tubes. Vortex the tubes for 10 seconds to mix the components.
Then incubate the tubes at room temperature for 30 minutes for the host E.coli to absorb the phages. Next, add 2.5 milliliters of molten TB1 top agar. Maintain that 55 degree Celsius to the appropriate titering and screening tubes.
After adding, immediately vortex the tubes gently. Now, pour the TB1 agar to the appropriate titer or screening TB1 agar plate. Allow the plates to stand for 15 to 30 minutes at room temperature.
Once the agar has solidified, invert and leave all the 10 screening plates in the stationary incubator at 24 degrees Celsius for 46 to 48 hours. Leave the remaining six titer plates in a stationary incubator at 37 degrees Celsius for overnight or a day. Following the incubation at 37 degrees Celsius, count the number of plaques formed in all the 3 titer 20 and titer 100 plates.
Following the incubation at 24 degrees Celsius, count the number of plaques formed in the 10 screening plates. To verify the putitive mutant plaques, core the plaque with a sterile wide bore pipette tip. Then transfer the plaque to sterile micro-centrifuge tube filled with 500 microliters of sterile SM buffer.
Then incubate the micro-centrifuge tube at room temperature for at least two hours at room temperature. Next, mix one microliter of the cored phage solution with 200 microliters of the G1250 plating culture in a sterile round bottom tube. Then incubate the mixture at room temperature for 30 minutes.
After 30 minutes, plate the cored phage solution in 2.5 milliliters of molten TB1 top agar and incubate at 24 degrees Celsius for 46 to 48 hours. Once the secondary plaques are visible, use a pipette tip to pick a single well-isolated Lambda cII mutant plaque. Transfer the plaque to a micro-centrifuge tube filled with 25 microliters of double distilled water in pipette several times.
Leave the tube on boiling water for five minutes. Then centrifuge the tube at 18, 000 times G for three minutes at room temperature. Immediately after centrifugation, transfer 10 microliters of the supernatant to a new micro-centrifuge tube containing 40 microliters of the PCR master mix.
After PCR amplification, purify the 432 base para-amplified product with the cII gene and its flanking regions incorporated using the PCR purification kit. Then sequence the cII transgene using a DNA analyzer to detect the mutations. The plaques obtained in both the titer 20 and the titer 100 plates are clearly visible by holding them next to a white light box and against a dark background.
Here, the bar plot depicts the mutagenic potency of various chemicals in the physical compounds tested. This is done by analyzing the extent of increase in the relative cII mutant frequency isolated from the mouse embryonic fiber blasts. The bar plot depicts all these test reagents showing statistically significant fold increase in the cII mutant frequency.
Next, the mutation spectra of the cII transgene in the mouse embryonic fiber blasts irradiated with UVB is demonstrated. The pie chart shows a significant increase in the relative frequency of single or tandem cytidine to thymidine transition at pyrimidine dinucleotides when compared to the control. Once mastered, this technique can be done in eight to ten hours if it is performed properly, excluding preparation times for bacterial streak plate in culturing.
In DNA sequencing, subsequent to scoring the cII mutants. After watching this video, you should have a good understanding of how to perform mutation analysis of a test compound in an in vitro model system.
