model organisms and genetic technologies Flashcards
Forward Vs Reverse genetics
Forward 1. Phenotype 2. Genotype 3. Biochemical function Reverse 1. New identified gene 2. Change genotype New phenotype suggests function of the gene
orthologue vs paralogue
Orthologues: homologues gene found in different species.
Paralogues: homologues gene found in same species.
Human and Mouse
- Placental mammals
- Adaptive immune system
- Similar anatomy
- Nervous system
Metabolism
human and zebra fish
- Vertebrates
- Nervous system
- Adaptive immune system
- Embryo development
Circulatory system
human and worms and flies
- Bilateria clade
- Blastocyst formation
- Motile
- 3 germ layers
- Innate immune
Cell-cell signalling
human and yeast
- Eukaryote
- Similar organelle comp
- Similar metabolism
Transcription / translation / gene regulation / cell cycle
Things to consider
- Biological question
- Feasible
○ Money
○ time ( gen time, n/o progeny etc.)
○ Measuring pheno
○ Homologous genes - Ethical?
- Genetically manipulated?
- Funded?
Translatable to humans
- Feasible
Good vs Bad - mouse
- GOOD ○ 90% genes syntenic (occurring on same chromosome) ○ 40% nucleotidally same ○ High relevance ○ 80,000 snp identified - BAD ○ Expensive ○ Ethical ○ Non-transparent Low progeny number and time consuming
Good vs Bad - zebrafish
- GOOD ○ Transparent ○ Behavioural studies ○ Faster replication - BAD ○ Less translatable ○ Ethical Specific expensive infrastructure (water tanks)
Good vs Bad - worm and flies
- GOOD ○ Cheap, fast ○ No ethics ○ Huge range of genetic tools - BAD ○ No cryo storage(FLIES) ○ Small amount of tissue ○ Non-vertebrate No adaptive immune
Good vs Bad - yeast
- GOOD ○ Cheap, fast ○ Cryo easy ○ Manipulation easy - BAD ○ distant Limited to single cell phenotypes
random vs targeted mutagenesis
Random mutagenesis forward
Targeted mutagenesis forward and reverse
Gene knockdown with RNAi
- Double stranded DNA produced (exogenous or endogenous)
- Dicer will take dsDNA and process it into short siRNA’s
- Associate with RISC and AGO complexes
a. Make a single stranded short mRNA
b. Bind via complementation to mRNA present in cell
Inhibition via degrading or inhibiting translation
Gene knockdown with RNAi good vs bad
Good - Target all genes in genome - Target specific genes in specific cells - Control temporally - Variable knockdown efficiency ○ Avoid lethality BAD - No 100% knockdown efficiency - Off-target affects
Gene knockout with homologous recombination
- Vector of choice with resistance (neoR) to something
- Recombination and expose to (neoR) killing the other gene
- Inject into ES cell for pregnancy
- Half of F1 will be het (+/-) and half homo dom (+/+)
Breed het F1 to make homo rec (-/-)
Gene knockout with positive/negative selection avoids random integration
- Regions allow recombination, PSM between homologous region, NSM outside homologous region
- Crossover in homologous region
- Target gene replaced with yellow construct with PSM
- In homologues recombination NSM removed
- In random integration NSM is included
a. Target gene not gone but somewhere else
b. With NSM we can differentiate between desired and undesired
Conditional gene knockout with the Cre / LoxP system
- Gene knockout in specific cell types / tissues
- Ends of target construct with loxP (sequences of DNA)
- Add Cre recombinase = homologous recombination
- If SAME orientation - intervening piece of DNA is excised
If INVERSE orientation - intervening DNA is reversed
IF one homo non-floxed mouse mates with a homo floxed mouse offspring are het for gene knockout
IF one het mouse mates with homo floxed mouse offspring are homo floxed
Spatial and temporal specificity with Cre / LoxP system
- One cell type specific promotor and one floxed gene
- ERt2 is bound to Cre
- Normal situation (left) 2 types of cells
○ Cells not in the liver i.e. not expressing Cre
○ Cells in liver expressing Cre but is fused with ERt2 (Cre is expressed but not active) - Tamoxifen injection (right)
○ Tamoxifen binds to the ERt2 complex activating Cre - promoting homologous recombination - gene of interest inactivated
We can control where it is knocked out (liver cells) and when (time of injection)
Gene knockout by CRISPR / Cas9
- Cas genes acquire viral DNA sequence
- Crispr repeats between spacer repeats (sequence the cell wants to target (viral DNA)
- CRISPR array transcribed into ssmRNA
- Cas protein processes array into individual crRNA
- crRNA used as guiding mechanism for cas protein
- Virus injects DNA - Cas protein recognises sequence by short sequence and then is cleaved
Non-homologous end joining vs Homology-directed repair
Non-homologous end joining
1. Site-specific DNA break
2. Random nucleotide deletion / insertion
3. Gene disruption
Homology-directed repair
1. Site specific DNA break
2. Added donor DNA with regions of homology
3. Homologous recombination to repair
Can change point mutation to corrected nucleotide
Good vs bad - CRISPR / Cas9
- good ○ Target any gene in genome ○ Quick, cheap ○ Works in all organisms ○ Specific cells/tissues ○ Control temporally / spatially - Bad ○ Not always desired alteration Off-target effects
Invertebrate gene manipulation
WORMS
DNA can be injected into gonad and spread throughout DNA
FLIES
- Transgene carrying mutation injected embryo
- Cell budding
Incorporate in some or all germ line cells
Targeted gene manipulation in flies with The Gal4 UAS system
- One fly with GAL4 gene is mated with fly Gene x fly
- Enhancer can be used for tissue specificity (brain, liver etc.)
- Gal4 is able to interact with upstream activating sequencing -anything downstream of sequence of UAS is transcriptionally activated
- Can result in knockdown of specific gene or overexpression which can be used to determine the function of gene
- Target manipulation
○ GAL80ts can be used to bind to GAL4 inhibiting function BUT only at a specific temp
- Inducible GAL4
○ Use a version of GAL4 that has incorporated a binding site for a hormone
○ In presence of hormone GAL4 is active
Without hormone GAL 4 is inactivated
