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THE SMALL RNAs WORLD - Coggle Diagram
THE SMALL RNAs WORLD
Small RNAs can regulate gene expression and provide a system to study the gene function
▸ affect the mRNA stability and translation
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siRNA & miRNA
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miRNA are encoded by the miR genes
⁍ In plant are involved in the deveolpmental and physiological process
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LIN-14 and LIN-4
Lin-14 has different binding sites for Lin-4.
- With a Lin-4 loss of funciton mutant, Lin-14 expression stay high because is not regulated anymore
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RNAi
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is FAST and requires no large forward genetic screen or no T-DNa or transposons and it is an efficient silencing strategy.
➡︎ it can overcome redundancy problems and can knockout more genes at once
DISADVANTAGES ➜
⁍ knockdown levels might vary from 50-90%, rarely approach 100%
⁍ might need to try several siRna sequences before you get an efficient knockdown
Applications of RNAi
▸ RNAi can be used for crops to improve quality
▸ RNAi can be used to silence genes in specific tissues
▸ RNAi can be used to silence linked genes
▸ RNAi can be used for high-throughput approaches in model plants
Initial construct of RNAi in plants ➡︎ promoter, antisense part, spacer (now it is an intron) and the sense part (siRNa)
GATEWAY Technology
uses lambda (phage) recombinaiton system to facilitate the transfer of heterologous DNA sequences between vectors.
based on the site-specific recombination system used by phage I to integrate its DNA in the E.Coli chromosome :
➜both organism have specific recombination sites, attP and attB and it is catalyzed by two enzymes Int and IHF
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2 step process:
- Cloning one gene of interest into the Entry Vecor using the BP reaction
- Subcloning the gene of interest from the Entry Clone to a destination vector using LR reaction producing the Expression clone
advantages⇾
- fast
- no use of restriction enzymes
- once you have made an entry clone the gene of interest can be easily sub-cloned into a wide variety of destination vectors using the LR reaction
Artificial miRNA
to design a functioning artificial miRNA you subimit your gene and you get to design a primer. The primers are used for a PCR which amplifies an endogenous miRNA from a vector
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