Functional genome Flashcards
The functional genome
- 90% research use non animal methods
- Cells behave differently in vitro to in vivo
- Most of the medicines we have today come from animal research
- Contributed to 70% of the nobel prizes
- Animal ill-health
- Research scientist seek to alleviate pain and suffering … rigorous HO monitoring
Mice
1 yr = 30 human years
- small reproduce quickly easy to handle and transport
more
- genetical similar to humans
lots of mouse strains and models already exist
- modern genomic engineering have allowed for a precise mutation to recreate a disease
Cripr/Cas9
- Clustered regularly interspaced short palindromic repeats (CRISPR
- CRISPR associated protein 9 (Cas9
• Bacterial adaptive immune system
-Protospacer (Target sequence of guide RNA - Protospacer Adjacent
Guide RNA binds to strand of genomic DNA, Cas9 endonuclease binds to non protospacer portion of gRNA + PAM of DNA
Mutations can be introduced through NHEJ and HDR
RNA rescue experiments : proving pathogenesis
Autosomal recessive cerebellar ataxias (ARCAs): neurodegenerative disorder….WES identified mutation in CHP1
WES and WGS identify candidate causative genes…functional follow up experiments required for proof
WES and WGS identify candidate causative genes…functional follow up experiments required for proof
Animal models represent an essential tool for disease aetiology, however can be costly and strict regulations apply.
Reality: a mix of in vitro and in vivo data contribute to convincing functional validation of gene variants
WES & WGS
- Rapid modern methods for high throughput DNA sequencing
- Whole Exome Sequencong (WES) used to capture the sequence of the coding region of the genome
- Whole Genome sequencing (WGS) captures the whole thing, not always needed
- Aim to identify potential disease causing genetic variants: personalised medicine
Bioinformatics: canididate gene filtering using WES
Bioinformatics: Candidate gene filtering using WES
15-20,000 coding SNPs reduced to one or several candidate genes.
Checked for co-segregation (family members) and validated by Sanger sequencing.
Functional evidence
- How does change affect protein behaviour?
- Detection of proteins in patient samples
- Tissue/Cell expression
- Development of in vitro and in vivo models for dysfunction of GOI
- How does the change affect cell or development of tissues?
- Identification of the molecular mechanism of action: common pathways!
Cell culture techniques (in vitro)
- Removal of cells from an animal and subsequent growth in favourable conditions.
- Primary cells have finite divisions but can immortalised to provide continuous source
- Provides a cheap, rapid and reproducible model for studying the normal physiology and biochemistry of cells
- A good alternative to using animal models, reducing the numbers of animals being used in research, less restrictions.
- Many tissue specific cell lines commercially available
Gene knockdown
- Based on endogenous microRNA gene silencing
- Modified to include GOI complementary sequence
- Package in DNA plasmid, expression controlled by a RNA polymerase 3 promoter
- 50-70nt. Exists nucleus, cleaved by a nuclease called Dicer (cytoplasm)
- Cleaved segments bind to RNA induced silencing complex (RISC) and direct cleavage and degration of complementary mRNA
- Short interfering RNA (siRNA): similar to ShRNA, chemically synthesised, not vector based
Where is GOI encoded protein localised?
Antibody staining
- Protein of interest
- Downstream target
Transfer cells with GFP tagged GOI (CMV promoter)
Why cell culture is not enough?
Cells behave differently in a petri dish/ flas to how they behave in a whole organism. 2D environment
Does not stimulate the actual conditions inside an organism. Signals from other tissues
No information about gene expression and function, with regards to developmental phenotypes
