In my recent work i have over expressed a gene using 35s promoter with BASTA resistant marker. surprisingly i am getting two different phenotypes for the transgenic line after BASTA screening.
I have got two individual lines showing Dwarf phenotype and 3 lines showing similar phenotype to that of WT. All these lines were confirmed using real time PCR and are showing over expression. interestingly the lines showing dwarf phenotype shows 1000 fold induction while the other ones are showing 100 to 200 fold induction in the transcript abundance. now the question arises that which lines to be chosen for further study?
what is the transgene and what is the aim of the experiment ? Give some more details
The transgenic is an over expression line for my gene of unknown function i am trying to characterize in arabidopsis. during the screening for homozygous line i used 5 individual T1 lines out of which 2 were dwarf and 3 were similar to WT but the fold induction in dwarf lines were 1000 times whereas 100 to 200 fold for other lines
Naturally you should select those plants which show altered phenotype (dwarf) for further analysis. You can search expression / RNA-seq databases with the gene to find its expression pattern and possible pathway of action
Generally we use individual lines to confirm that the phenotype is due to over expression and then validate using real time PCR. as i have told earlier that all the 5 individual lines are showing over expression (checked by real time PCR). but the phenotypic difference between these lines is confusing.
How can we rule out the fact that the phenotype which i am getting is due to over expression and not due to disruption of some other gene due to T-DNA insertion ???
It's strange that two independent insertion lines showed the same phenotype. But of course you can't rule out the the insertion point as a cause, unless you sequence the insertion region to find it out. It looks like a dose effect. Some ideas: Maybe you can check the levels of your protein in the 5 lines to check if translation is affected. What about KO mutants of your gene??? If your gene is regulating plant size the mutants should have the opposite phenotype.
Thanks @Tamara Hernández-Verdeja for your response.
Firstly, The gene has another homolog so i could not find out any obvious phenotype in homozygous TDNA knock down line. Secondly, the TDNA knockdown line has an insertion in the 3' UTR region of the gene. And thirdly with recent sequencing data generated by TAIR, i came to know that there is polymorphism in the salk line which i am studying. Presently i am also trying to clean those polymorphism by backcrossing with WT, in the due course of time i am analysing the over expression line to get the insight into the function of the gene. hope my approach is right:)
Hello again,
It seems you are working with a tricky gene ;) That T-DNA line with the insertion in the 3'UTR doesn't look good. If I were you I would try to generate KD lines, iRNA, amiRNA...
Returning to the over expressor lines you have to make a difficult choice unless you want or can continue working with all the lines until you have any clue. What I would try: check a few more independent lines in case there's a relation between expression levels and size or to unbalance the 2vs3, check for other phenotypes in the lines based in why you choose this gene.
First of all if the same phenotype is coming from independent lines, then it is most likely gene specific. It is very normal to see different phenotype in T1 lines, not all lines show same phenotype, BASTA resistance has nothing to do with it. To confirm whether the phenotype is coming from your GOI, you can do further loss-of-function analysis and similar other experiments. If you have any putative target of your gene, look for its expression too. Looking at the expression data, it is correlated with your phenotype, so it is good. If your gene has a paralog, make a double mutant, dont worry about backcrossing, it is wastage of time. you can prove it later by complementation. make a double mutant. Some genes only show phenotype when overexpressed, not otherwise. 3' UTR T-DNA may not work.
Based on your observation, it seems two T1s with dwarfism look dominant, but you should check T2s confirming 3:1 segregation of dwarf phenotype. The first thing is to generate stable T3 lines that you can apply tools that people mentioned above.
Hi!
If I were you after genotyping to single gene copy I will check again phenotype and study both. They are certainly different events!
Good luck!
These two events are likely to be because of insertion of the transgene in two distinct insertional events. Are these events single copy insertions?
Thanks for all the valuable responses
We have not checked the single copy insertion.....generally in Arabidopsis we study 3:1 segregation for homozygous selection. I will try to first check the copy no. as per your suggestions.
I would choose those lines with highest fold of induction; it is already great that you can correlate the transcipt levels with the phenotype. You can comment on your observation in your future paper.
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