Encyclopedia of Experiments: Biology
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Transcript
- When injected embryos develop into G0 adults, cross all individual flies to suitable balancer stocks to expand potential F1 CRISPR edited lines and be able to track the inheritance of the genomic modification. Then, cross randomly chosen F1 single males to suitable balancer females to be able to recover offspring if an F1 male is positive for the mutation. Once the cross has taken, transfer the F1 male to a tube for screening.
To extract genomic DNA, squish the fly with a pipette tip containing extraction buffer. Once the tissue is broken down, eject the liquid. The extraction buffer contains proteinase K, an enzyme that digests proteins in the sample and is inactivated by high heat.
Now that DNA is accessible, proceed to PCR screening. For example, use primers designed to amplify a region that includes the new sequence. Only in the presence of the modification will the second primer bind and a PCR product be amplified. In the example, we will see flies being bred and screened by PCR for insertion of a transactivation sequence replacing the first exon of the branchless gene.
- When nos-Cas9 embryos previously injected with both the guide RNA expression plasmid and the replacement donor develop into adults, cross G0 flies individually to balancer flies from a line appropriate for the targeted allele. Anesthetize the F1 offspring from each G0 cross on a carbon dioxide pad and randomly pick 10 to 20 males under a stereo microscope. Individually cross each selected male to a balancer female from a line appropriate for the targeted allele.
When the F2 larvae hatch, pick the F1 father from each cross. Extract genomic DNA by squishing each fly for 10 to 15 seconds with a pipette tip containing 50 microliters of squishing buffer without dispensing the buffer. Then, dispense the remaining buffer into the tube and mix well.
Incubate at 37 degrees Celsius for 20 to 30 minutes. Following the incubation, place the tubes in 95 degrees Celsius heat block for 1 to 2 minutes to inactivate the proteinase K. After spinning down for 5 minutes at 10,000 times g, store the preparation at 4 degrees Celsius for further PCR analysis.
Perform three-step PCR based screens using primers forward 1 and reverse 1 to screen for the existence of the insertion or replacement. Perform PCR using forward 2 and reverse 2 primers to verify the insertion or replacement from three prime region. Perform PCR using primers M13F and reverse 3 to check ends-in HDR.
- Keep the fly lines with the confirmed ends-out HDR and establish balance stocks from the F2 generation. Out cross the balancer flies again to remove any unintended mutations on other chromosomes.
