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THE SMALL RNAs WORLD - Coggle Diagram
THE SMALL RNAs WORLD
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RNAi
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Disadvantages: knockdown level may vary from 50-90%, but rarely approach 100%; also, you may need to try several siRNA sequences before you get efficient knockdown.
Classical construct: promoter, antisense part, a space, the sense part
Gateway Cloning Technology: uses the lambda recombination system to facilitate transfer of heterologous DNA sequences (flanked by modified att sites) between vectors.
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Recombination process
- The phage I encoded protein Int (Integrase) and the E.coli protein IHF (Integration Host Factor)
- Upon integration, the recombination between attB and attP sites generate attL and attR sites that flank the integrated phage I DNA.
Excision process
- Process catalyzed by Int and IHF in combination with the phage I protein Xis
- attL and attR sites surrounding the inserted phage DNA recombine site-specifically during the excision event to reform the attP and attB sites.
att1 and att2 sites
attB1 only reacts with attP1, attL1 only reacts with attR1 (same the att2)
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Artificial miRNA
Reduce the a-specific silencing off-target genes and can be designed in order to recognize YFG or a family of genes.