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 Gene Gun and Biolistics 
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Joined: Fri Oct 08, 2010 6:08 am
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Post Gene Gun and Biolistics
Hi
I wanted some more information on gene guns and biolistics from other than wikipedia. Is the preparation of genes for a gene gun different for plants and animals?

Thanks


Fri Oct 08, 2010 6:23 am
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Joined: Thu Aug 19, 2010 3:18 am
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Post Re: Gene Gun and Biolistics
Yes, one example would be that your gene must contain a promoter sequence that is suitable for the organism its being inserted into. A gene with a plant promoter will not function properly in a mammal.


Sat Oct 09, 2010 12:35 am
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Post Re: Gene Gun and Biolistics
Thanks.

One more question: I was wondering whether you still have to use a plasmid vector from agrobacterium if you are using plant cells. My understanding was that the genes are "shot" into a callus of undifferentiated cells, and then are grown into transgenic plants from those cells.


Sat Oct 09, 2010 5:50 pm
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Post Re: Gene Gun and Biolistics
No, a binary vector containing T-DNA borders are not necessary. Those are only important for Agrobacterium mediated plant transformation because Agrobacterium proteins like VirD2 bind to those regions.

However, vectors are usually linearized prior to biolisitc transformation. In facts its common to simply cut out the gene cassette and removing the vector backbone.

Also, you will need a plasmid containing a suitable selection marker. Otherwise you will have some difficulty selecting for your positive transformants.


Sat Oct 09, 2010 7:47 pm
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Post Re: Gene Gun and Biolistics
whoa...I need simple english. what is a postive transformant? gene cassete? T-dna?
thanks


Mon Oct 11, 2010 4:11 am
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Post Re: Gene Gun and Biolistics
Ok.

Transformation = is the process of inserting DNA by a cell/organism and the expression of that Gene.

So a positive transformant is a positive result of the transformation process. So if you do plant transformation and 1% actually take up the DNA, insert it, and stably express that gene, then that 1% are positive transformants.

Gene Cassette. How familiar are you with plasmids? With the structure of vectors? Your typical bacterial plasmid has several features. For instance it has an origin of replication (Ori) where replication of that plasmid is initiated. Depending on what Ori is present, the plasmid will be high-copy number or low-copy number. There is usually a selection marker, like ampicillin resistance. There is usually a multiple cloning site. So you have all these features of your typical plasmid vector that form the backbone of the vector. The gene cassette then is your gene, promoters, terminators that you clone into the vector.

T-DNA. This refers to Transfer DNA. Its the DNA that is inserted into a plant by Agrobacterium. In wild Agrobacterium, it contains genes that co-opt control of the plant. T-DNA has specific conserved sequences on each end that are recognized by Agrobacterium proteins. These sequences are essential for Agrobacterium transformation to work. Binary vectors used for Agrobacterium plant transformation contain just these border sequences. You clone your gene of interest in between them, so that only your gene of interest gets inserted.

Can I ask something? Are you asking this because you want to attempt Biolistic Plant Transformation? Let me say that this is not an easy task. For well established species, the best labs get ~15% efficiency. This is not a realistic starting place for people wanting to start in on biotech.


Mon Oct 11, 2010 4:34 pm
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Post Re: Gene Gun and Biolistics
Thanks.

Where do you think is a realistic starting point for people interested in genetic engineering?


Mon Oct 11, 2010 5:26 pm
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Post Re: Gene Gun and Biolistics
E. coli. Those who do plant transformation use E. coli as an intermediary. Really all those in biotech use E. coli as an intermediary because its simple to work with and the tools are well established. If you want another organism to work with, that utilizes many of the same tools, then try Yeast. But even then, E. coli will be necessary for initial cloning.


Mon Oct 11, 2010 6:57 pm
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Post Re: Gene Gun and Biolistics
Okay. I thought that biolistics would be the simplest place to start, but I guess I was wrong. Thanks for the information.


Mon Oct 11, 2010 7:05 pm
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