Future research area for CRISPR/Cas9 in wheat?

[Dean22April]

So I am in a group testing whether the plants we have of a certain wheat line are expressing the Cas9 transgene and if the gene of interest that we've tried to target has been edited by CRISPR/Cas9.

So far I have: -Extracted DNA and run a PCR and gel electrophoresis to confirm some (around 75%) of the plants have at least the starting fragment of the Cas9 gene
-Extracted RNA and run an RT-PCR and gel electrophoresis to confirm some of those have the mRNA for the Cas9 (so are expressing the DNA) - around half were 
-Extracted and run DNA for the guide RNA (which all showed to be expressing worryingly) 
-Quantified the amount and quality of our mRNA using a bioanalyser 
-Sequenced the guide RNA and some of the Cas9 DNA samples (which we'll get the data for tomorrow).

So now we only have 3 weeks left in the lab and we need to decide what to do, whether we go back and re-do some processes or check for expression of proteins or editing of the gene. We are definitely planning to test whether the gene itself has been edited, but don't know what else would be useful. Any ideas what direction we should pursue?
Thanks!!

[Vmedvil]

On 10/26/2017 at 0:55 PM, Dean22April said:

So I am in a group testing whether the plants we have of a certain wheat line are expressing the Cas9 transgene and if the gene of interest that we've tried to target has been edited by CRISPR/Cas9.

So far I have: -Extracted DNA and run a PCR and gel electrophoresis to confirm some (around 75%) of the plants have at least the starting fragment of the Cas9 gene
-Extracted RNA and run an RT-PCR and gel electrophoresis to confirm some of those have the mRNA for the Cas9 (so are expressing the DNA) - around half were 
-Extracted and run DNA for the guide RNA (which all showed to be expressing worryingly) 
-Quantified the amount and quality of our mRNA using a bioanalyser 
-Sequenced the guide RNA and some of the Cas9 DNA samples (which we'll get the data for tomorrow).

So now we only have 3 weeks left in the lab and we need to decide what to do, whether we go back and re-do some processes or check for expression of proteins or editing of the gene. We are definitely planning to test whether the gene itself has been edited, but don't know what else would be useful. Any ideas what direction we should pursue?
Thanks!!

Insert the gene Via a constructed non- reproducing Plant Lentivirus or Lentiviral Vector is what I would do, but that would take longer than 3 weeks to complete, they are very efficient at sending DNA into cells, you would have a 99% or higher plant expression of the gene, I dunno at this point unless you have a Oligonucleotide synthesizer much better than mine at a cycle of 5 minutes. 

Edited November 13, 2017 by Vmedvil

[Dean22April]

On 13/11/2017 at 6:59 AM, Vmedvil said:

Insert the gene Via a constructed non- reproducing Plant Lentivirus or Lentiviral Vector is what I would do, but that would take longer than 3 weeks to complete, they are very efficient at sending DNA into cells, you would have a 99% or higher plant expression of the gene, I dunno at this point unless you have a Oligonucleotide synthesizer much better than mine at a cycle of 5 minutes. 

Thanks so much Vmedvil!!

[Vmedvil]

6 minutes ago, Dean22April said:

Thanks so much Vmedvil!!

Yes, just make sure that your virus that is used as a base for the vector is lysogenic and not lytic in its DNA machinery otherwise it won't work.

Edited November 27, 2017 by Vmedvil