Hello everyone,
Using CRISPR CAS9, I got homozygous +1 mutants for a rice gene. The CAS9 target lies in the middle of the gene. After mutation there is no stop codon in gene body. Only one in UTR region is observed. Now my question is that this gene will normally be transcribed (so I can not use RT PCR to confirm the knock out). Also as the newly observed stop codon lies a little ahead of the original stop codon in UTR region (So I presume that we cant not use western to confirm knockout). I have just sown the plants and am looking to observe some phenotype associated with mutation. Your suggestions regarding confirming the knockout of the gene will be highly appreciated. Thanks!
I'm a bit confused why you think you can't use either method as those are both used to confirm the lack of gene product (mRNA and/or protein). You can still check expression by qPCR. Sometimes the missense mutation is enough to activate mRNA degrading pathway. You should also check protein expression by western blot if you have the appropriate antibodies.
If I were you, I would not call your mutation a "knockout" unless you have evidence to demonstrate a lack of gene product. Simply call it a mutant line for now.
Dear Katie A Burnette ,
Thank you very much for your answer. Your suggestions are always helpful. I will follow accordingly.
How did you reveal the +1 mutation in your gene? Via DNA sequencing? You can do the same for mRNA, just prepare the cDNA, run PCR to amplify gene fragment surrounding the potential mutation and sequnce the PCR product.
If you have appropriate antibodies, you should try WB as this would show you immediately whether there is still some protein or not. Regardless of the stop codon availability, +1 mutation will entirely change the reading frame, so no protein should be detected (or some truncated fragments depending on the antibodies epitop).
Dear Eugen Werwein,
The mutation is in reading frame, i know it will be transcribed into mRNA. But my question was whether that was loss of function for the gene or not as it does not lead to premature stop codon and only one in UTR region (little ahead of the original stop codon) is observed. Further to add that due to frame-shift an important characteristic domain of the protein is completely destroyed.
I will try WB as recommended by you and others. Thanks.
I would agree with some of previous suggestions. Although it did not introduce any internal stop codon, if the +1 mutant did happen, then it would change the amino acid residues translated from the downstream of +1 insertion. If that happens, it would definitely change the polypeptide properties obtained from the mutant gene. Therefore, you might be able to detect the differences using the protein detection protocol.
Moreover, the +1 insertion should also create an SNP in the mutated position. You may use a simple PCR SNAP (single nucleotide amplified polymorphirm) detection methods to differentiate the mutant vs. the wildtype. Create two sets of SNAP specific primers to amplify alternative amplicons and determine whether you have either wild type or mutant homozygous or heterozygous individual base on the generated amplicons. I will be happy to share the procedure with you if you want it. Good luck...
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics