Term
| Give examples of natural and experimental gene transfer |
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Definition
Natural: sexual reproduction, parasites, symbionts, transposons, uptake of DNA in solution
Experimental: Biological vectors (viruses, agrobacterium), direct gene transfer (chemical, electrical or mechanical treatments to breach membrane barriers) |
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Term
| All methods of genetic transformation in plants only transform individual cells, true or false? |
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Definition
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Term
| What are the general steps in plant transformation? |
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Definition
Transfer DNA into cells
Select transformed cells (kill rest, selectable marker genes)
Regenerate a whole plant from a single cell
Verify transformation |
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Term
| Why is agrobacterium a useful vector in genetic modification? |
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Definition
| Part of its Ti plasmid DNA is transferred into plant cells (T-DNA) |
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Term
| Describe the structure of T-DNA in the Ti plasmid |
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Definition
Flanked by left and right border sequences
Has phytohormone synthesis gene
Has opine biosynthesis genes (nutrient secretion for Agro) |
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Term
| What steps are involved with inserting target genes into a plant via agrobacterium? |
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Definition
Cut out disease producing genes
Insert DNA you want to transfer
Infect plant tissue |
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Term
| What is a technical problem using agrobacterium in genetic manipulation? How is it overcome? |
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Definition
| Ti plasmid is too big, breaks often. Overcome with binary vectors (splitting plasmid into two smaller plasmid, one with the genes neccessary for DNA transfer and theother with the T-DNA border sequences and a good polylinker |
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Term
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Definition
| A site on DNA for restriction enzymes |
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Term
| Why do researchers always transfer two genes with agrobacterium? |
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Definition
| Need a selectable marker gene (antibiotic resistance) to confirm genes have indeed been taken up |
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Term
| What are advantages to using agrobacterium over a gene gun? |
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Definition
| Gene gun can make a mess, don't know what mutations you're getting. Agro can put 2 copies in gene instead of one and has a higher level of specificity |
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Term
| What would be the general steps to doing a transformation using agrobacterium? |
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Definition
Prepare tissue in culture
Prepare agrobacterium culture
Co-cultivate with agro (with engineered T-DNA)
Culture tissue with antibiotics (to select transformed plant cells and kill agrobacterium) |
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Term
| What is the main limitation of using agrobacterium in transformation? |
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Definition
| Host range, does not efficiently infect monocot plants |
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Term
| Describe two methods of direct gene transfer and when to use them |
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Definition
Electroporation: use with protoplasts
Gene gun: gets through wall of intact cells |
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