ENVB 437 Lecture 21: Eukaryotic cells and viruses – lecture 21 (March 28th 2018)
4 Jun 2018
School
Department
Course
Professor
Eukaryotic cells and viruses – lecture 21 (March 28th 2018)
Generation of siRNA
- If youre ordering RNA, it is very labile so if it gets contaminated you will lose all the siRNA
o If you have it in the DNA form you can make it any time – more stable
Slide 46-48
- To apply it to mammals was a little more difficult than in plants
o Because of the non-specific silencing pathway
▪ If you introduce dsRNA >30 nucleotides, that induces a global inhibition of DNA expression
on the cell
• Can do in plants, Drosophila, etc
o You shut down protein synthesis and inactivate the synthase molecules as well as RNAses and all the
RNA
- Rather than using dsRNA, you have short interfering RNA sequences that you introduce in the cell and KO a
particular transcript
o A typically good siRNA would be 70-80% of knowing out that transcript
▪ Contrary to when you KO a gene completely
- How can we get it into a cell?
Slide 50
- They can be engineered such that the cell line has a contrast that is incorporated directly in the genome and
expressed from the chromosomal location
o Inducible systems can be turned on or off as much as you want
▪ The inducer can be added to the medium and you can observe the effects before and after
o Can also make KO mice, they are easy to work with in terms of making transgenics
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Eukaryotic cells and viruses lecture 21 (march 28th 2018) If youre ordering rna, it is very labile so if it gets contaminated you will lose all the sirna. If you have it in the dna form you can make it any time more stable. To apply it to mammals was a little more difficult than in plants: because of the non-specific silencing pathway. They can be engineered such that the cell line has a contrast that is incorporated directly in the genome and expressed from the chromosomal location. When the hairpin is introduces, the hairpin will be clipped. Or you can drive synthesis in opposite directions and directly make rna. Using a single promoter, you drive transcription in one direction. In the design process, you make a spacer that will be a loop and also make an antisense: so that when transcription occurs you make a short piece of rna that would fold back onto itself.
