How do I produce homozygous transgenic lines harboring a single copy of a transgene using BASTA selection?
Dear Akila,
Check this out and ask the authors here on RG:
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.321.3163&rep=rep1&type=pdf
You cannot know or control a piori how many copies you'll have. Anyway you should be looking for T2 with a segregation ratio Basta R: Basta S of 3:1 (use a high number of plants for this purpose and do Chi-squared test). Transfer on soil several Basta-resistant plants of 'good' lines, collect seeds from each plant and put the T3 on Basta again (now you should find homo).
To be 100% sure of the copy number you should do a Southern blot.
Otherwise (or even better in addition), work a least with 3 INDEPENDENT lines to avoid phenotypes coming from multiple insertions.
1. You cannot distinguish 'homozygous' transgenic lines from 'heterozygous' in a mix population of +/+ and +/- transgenic lines by using selection (BASTA in your case). Both (+/+ and +/-) should survive BASTA selection.
2. If you have a 'single-copy' primary transgenic line (as you stated), propagate it into next generation. In the progeny, you will have +/+ (homozygous) and +/- (heterozygous) genotypes for the transgene. You can check homo- or heterozygous lines by quantitative PCR or by progeny test (need another generation).
Thank you Prof. Béatrice, Dr. Roberta and Dr. Yuan-Yeu Yau :)
To Dr. Roberta and Dr. Yuan-Yeu Yau;
If I'd grow plants till T3 generations, by using BASTA how would like do the selections of homozygous seedlings.
Any explainable illustrations with Mendelian genetics available?
Thanks a lot :)
http://www.slideshare.net/Pammy98/mendelian-genetics-ppt
There are many power points on this topic on google.
Hi Akila,
Regarding your last post---
If you only want to use selection (BASTA) method to find out homozygous plants. You can use 'progeny test'. This is what you do:
1. You have a single-copy primary transgenic line (T1, +/-, hemizygous).
2. Self-pollinate this plant (if Arabidopsis) to produce seeds. These seeds contain +/+, +/- and -/- genotypes for the transgene.
3. Selecting those seeds under BASTA, you will get some survived plantlets (T2). Within the survived plants, some genotype are +/+ and some are +/-. Ratio should be 1:2 for +/+: +/- . The -/- should not be survived.
4. Grow out some of those survived T2 plantlets.
5. Harvest seeds from those individual plantlets. Avoid seed cross-contamination between plants.
6. Do a BASTA selection on those seeds from different individual plantlets. You should observe some plates with all survived plantlets. They are all +/+, indicating that these seeds are derived from +/+ T2 plants.
7. Keep the seedlings (T3) from those plates. they are all +/+ (homozygous).
** This strategy can work only when the bar gene is not gene-silenced, for example, by methylation after several generations.
Hi Akila,
What plant species are you working on? Self-pollinated species or out-crossing species?
Dear Prof. Béatrice & Dr. Yuan-Yeu Yau,
Thank you very much
Dr. Yuan-Yeu Yau,
Thanks again for spending your valuable time on explanations. This is regarding arabidpsis.
Thanks again !!!
I don't want to reapeat those many ideas you received so far just giving a short addition.
If you could fish out a single copy, heterozygous plant (by any of the upper mentioned methods), than you can start special tissue culture - most often anther culture or microspore culture -, to produce doubled haploid (DH) plants, where all the nuclear genes will be homozygous, including your GOI.
But it is important to know what species you work with...
I agree with all above answers, in brief you need to carry over your transgenic plants up to T3 generation thereafter you can do southern blot to determine the copy number of independent lines.
You may follow a simple screen: Grow progeny of your T2 plants and spray with BASTA. You may even do a paste assay. If all plants show same response then you know your T2 is homozygous. If you are unable to recover one at T2, follow the same approach to T3 single plants and look at their progeny. Let me know if you would like additional details about the proposed approach. Good Luck!
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