EXOGENOUS APPLICATION OF RNAS TO DISCOVER GENE FUNCTION Flashcards
When can RNA silencing occur?
Even after gene silencing!
How is gene silencing achieved?
By a stable or transient expression of genetic constructs that produce hpRNA
hpRNA
hairpin RNA
How do we discover gene expression?
By inhibiting the gene expression at a post transcriptional level
Reverse genetics
A way to find out gene function; it is based on phenotypical changes that can occur when we over or underexposes a gene
Plants own PTGS
It helps the plant to protect against viruses
How to we use a Plants own PTGS?
- we let DCL splice the dsRNA into sRNA and miRNA (21-24 of them)
- We add Argonaute containing silencing effector complexes
- it guides a sequence specific mRNA cleavage
- the silencing pathway starts
How does exogenous applied dsRNA enter a plant cell?
It enters through wounded or abraded surfaces -> moves away from the initial point of application
What is the best cell type to silence something? Endogenous ones or transgenes?
Transgenes!
How does a silencing signal move inside a plant?
Via vascular tissues from “the root to the shoot”
Vascular tissues
Vascular tissue is comprised of the xylem and the phloem, the main transport systems of plants.
Stable transformation
Used for the stable introduction of a gene into a plant- meaning the gene will be fully integrated in the host genome, so it is expressed continuously, and will also be expressed in later generations of the plant.
How do we use a stable transformation to silence RNA
We use siRNA generated constructs that activate the RNA silencing pathway
What is the problem with choosing stable transformations too silence RNAi?
It is labour intensive and time consuming
Genetic engineering via biolistic method
regeneration and selection of stable trans genetic plants; molecular analysis of the plants trans genetic state;
phenotypical analysis of plant generations
