Dear All,
I have been trying to knock-in GFP in a particular locus at its N-terminus. I have prepared the Donor construct with a short arm of 0.8 kb and a long arm of 1.6 kb. I have been doing the same thing as explained in the following paper -
http://dev.biologists.org/content/141/19/3807.full
I have 4 samples, one is an uninjected control, only TALEN-injected embryos, only Donor construct (linearised) injected embryos and TALEN + Donor injected embryos. To test whether, the knock has been successful, I isolate RNA and synthesize cDNA. I don't use genomic DNA to check the integration, since even after 5 dpf, non-integrated DNA is still present in the larva which hinders the screening of the integration event. (my past experience). I perform RNA isolation kit by Qiagen. Then I use a set of primers that amplifies some part of GFP and exon 1. GFP is just N-terminus to the exon 1. Technically, I should get a PCR band only in TALEN+Donor injected embryos but I get a band of the same size in just donor construct injected embryos too. So, I am not sure whether my knock-in is successful. Technically, my RNA preparations should not have any donor construct left since I treat them with DNAse1 provided by the kit. I don't know if integration is possible without TALEN injections too. The results are quite confusing. Let me know if anyone of you has experienced such strange outcomes with TALEN-mediated Knock-In in zebrafish.
http://dev.biologists.org/content/141/19/3807.full
Hi Chaitanya you can get random integration of your plasmid into the genome but i would think its unlikely that you would intergrate the plasmid into the rna of interest and have it tanslated. Therefore i think you likely have some residual plasmid in your RNA. Have you thought about using DNA instead and using a fwd primer outside the short homology arm and a reverse primer within the gfp? Then you'll know if you get specific intergration.
Based on your description, it seems that if you would get a PCR band if you used your plasmid as the template. It is near impossible to troubleshoot: causes may vary from incomplete DNase digestion of your sample to contamination of any of the reagents used for RT or PCR. As Robert suggested above, use primers outside of your homology arms: genomic for regular PCR and next-exon for RT-PCR. Also, you did not mention if you see expected mosaic GFP fluorescence pattern ...
for KI screening, it is of importance to examine the KI events in the genome level first. I suggest you design specific primers on donor and genome, respectively. For KI in the zebrafish, our intron-mediated method is very powerful, and you can get the related KI elements from addgene (https://www.addgene.org/Jiu-lin_Du/). Till now, we have made 13 different KI lines in five genes, including lines with tagging cells and tagging endogenous proteins. Hope these help, good luck!
http://www.nature.com/cr/journal/v25/n5/full/cr201543a.html
http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path[]=4547&pubmed-linkout=1
https://www.addgene.org/Jiu-lin_Du/
hi all, it took me a lot of time to reply. iI did not see any GFP signal...but I expected that it will be a very weak signal...it's a transcription factor which is tightly regulated. So, as for the proof of principle, I have carried out this PCR. As suggested use of a primer outside GFP, it was not possible because, in the first place, amplification for the donor construct was not easy itself, hence we undertook this RNA approach. But, thanks a lot for your responses.
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