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C. Elegans
Drosophila
Zebrafish
Mice + Humans
Random
100
This is the technique you can use to determine which cells are involved in a given process in C. elegans
Cell ablation
100
What chromosome is the CyO balancer used for, and what are its main components?
2nd, recessive lethal, dominant visible marker (curly wings), scrambled order
100
What are some drawbacks of morpholinos?
Only effective for the first few days of development, off-target effects, toxicity
100
What does an odds ratio greater than 1 mean?
Increased frequency in cases
100
What is the term for the portion of individuals in a population that show a phenotype given that they have a specific genotype?
Penetrance
200
The screen you would perform for recessive lethal mutants in C. Elegans
F2 Clonal screen
200
If we mutagenize parents, why do we wait until F1 to look for a phenotype?
We want to obtain a fly that has the mutation all throughout - which can only be obtained from germ cells
200
When would you use CRISPR vs morpholinos?
Later vs earlier phenotypes, slower vs faster
200
How do you control temporal specificity in lineage tracing experiments?
Timing of tamoxifen pulses
200
Two genes (B and E) have an epistatic relationship. If the phenotype conferred by the ee allele always shows regardless of the genotype at the B locus, is E epistatic to B or B epistatic to E?
E is epistatic to B
300
How would you distinguish self-progeny from cross-progeny in C.elegans?
Include dominant marker in male or recessive marker in female
300
What will the phenotype be for each of the following? 1) UAS-GFP; Gal4 2) UAS-GFP; Gal80 3) UAS-GFP; Gal4; Gal80
1) GFP+ 2) GFP- 3) GFP-
300
How would you control for off-target effects with morpholinos?
CRISPR, inject MO into mutant, inject non-targeted, p53, multiple MOs
300
What is the threshold for genome-wide significance?
5 x 10^-8
300
How would you assess if siRNA knockdown was successful?
Western blot for protein levels
400
In pooled linkage analysis, what is the expected frequency of a non-causal SNP in the mutant population?
50%
400
Why do we scramble balancer order?
To suppress recombination - otherwise WT would eventually win out
400
When creating a CRISPR mutant, what stage do you inject at?
1-cell stage
400
What is the difference between IBD and IBS?
IBS implies that individuals have the same allele, but not necessarily because of inheritance from a common person
400
Are mutants that fail to complement on the same gene or different genes?
Same gene
500
How would you determine in which cells a gene acts?
Mosaic analysis
500
How would you find a dominant enhancer for a partial loss of function mutant?
Screen F1 for more severe phenotype
500
In an F3 mutagenesis screen, does each F1 fish have the same mutation?
No, each has a unique mutation
500
Outline the steps for knocking out a gene in a mouse
Homology arms (sequences homologous to target region) Positive selection (inside homology arms) - NeoR (G418 selection) Negative selection (outside homology arms) - Thymidine kinase or dipthera toxin Electroporate into mouse embryonic stem cells Select with G418 - the cells that survive have the target construct Could have correct insertion or could have random insertion with entire construct Add gencyclovir or DTA to get rid of random insertions Confirm via PCR, sequencing, or southern blot Inject these ES cells into blastocyst and transfer to pseudopregnant females Hosts have black coat - inject into inner cell mass Your cells have genes for a brown coat Chimeric offspring with brown and black coats x WT Screen F1s via PCR for mutant hets Raise, cross, and analyze
500
How are siRNAs and morpholinos different?
Morpholinos require > 15 bases of complementarity, siRNA only need 7. MO is steric occlusion – they do not degrade mRNA!, siRNA goes through RISC pathway. MO can also be used to modify splicing