Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes
-
Upload
bethesda-sharp -
Category
Documents
-
view
36 -
download
0
description
Transcript of Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes
![Page 1: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/1.jpg)
Analysis of gene function
Loss-of-function
Many gene knock-out have no obvious phenotypes
Redundancy?
No perfect redundancy => subtle phenotype
Gain-of-function
Many give observable phenotypes (e.g. oncogenes)
mis-expressionenhanced expressionectopic expression
constitutive activation
![Page 2: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/2.jpg)
Using Drosophila to analyze human gene function
Homologous gene in fly (FlyBase)
find fly mutation
study fly mutation
identify interacting genes
check in human for involvement of the interacting genes
Human gene family usually have more genes than in fly
easier to study in fly
can not address functional diversity
![Page 3: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/3.jpg)
-/-+/++/-
Clonal (mosaic) analysis
Analysis of gene with pleiotropic functions (e.g. at later stages)
Determine the site of gene function
![Page 4: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/4.jpg)
+
m
FRT
FRT
marker
hs-FLP
+
m
FRT
FRT
marker
+ marker
+FRT marker
+ marker
mFRT
m
HS
Making a mitotic clone
Random location
![Page 5: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/5.jpg)
loxP loxP
Enhancer-Cre recombinase
=> tissue-specific knock-out
![Page 6: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/6.jpg)
![Page 7: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/7.jpg)
No fly homolog
Ectopic expression in fly => phenotype
Determine what process/pathway is affected
Similar function in human?
Search for interacting genes in fly
Similar gene interaction in human?
![Page 8: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/8.jpg)
GAL4 X
UAS
GAL4
specific enhancer targeted expression of X
![Page 9: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/9.jpg)
CD2Actin promoter
FRT FRT
STOP GAL4
UAS
UAS
X
GFP
hs-FLP random location; can sample all cells
Enhancer-FLP
Clonal induction of expression
![Page 10: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/10.jpg)
enhancers Endogenous gene
UAS
UAS
Drives anti-sense expression
Drives expression
Cross with GAL4 lines => targeted expression
Targeted expression screen
![Page 11: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/11.jpg)
Flowchart for ectopic expression in fly
1. Make UAS-X construct
2. Microinjection to fly embryo
3. Isolate transformants and set up balanced lines
4. Drive expression with GAL4 lines GMR-GAL4 dpp-GAL4 Eq-GAL4
5. Examine phenotype what cell types are affected? what cellular processes are affected? fate transformation? cell death or proliferation?
![Page 12: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/12.jpg)
Using fly to study human gene function
No fly homolog
Misexpression in fly
Phenotype in fly
Function
Interacting genes
Fly homolog
Fly mutant
![Page 13: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/13.jpg)
The Drosophilacompound eye
![Page 14: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/14.jpg)
利用果蠅研究人類疾病
人類MJD基因表現於果蠅造成類似的神經退化壞死現象
正常的MJD基因 致病的MJD基因
![Page 15: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/15.jpg)
Eye-antenna
Leg
Wing
dpp-GAL4 + UAS-GFP (dpp>GFP)
![Page 16: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/16.jpg)
dpp>eyg dpp>ey dpp>eyg+ey
Coexpression of eyg and ey showSynergistic effect in eye induction
![Page 17: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/17.jpg)
wt gv (eyg) Eq>hth
Dorsal midline fusion of the thorax
![Page 18: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/18.jpg)
脊索閉鎖不全
![Page 19: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/19.jpg)
6. Predict interacting genes based on knowledge in fly study obtain relevant mutants test for genetic interactions
Modifier mutation (suppressor, enhancer)
Second site mutation that modifies the mutant phenotypeof the first gene
a-/a+ b-/b+ => mutant phenotype
a-/a+ b-/b- => stronger phenotype (dominant enhancer)
a-/a- b-/b- => synergistic (more than additive) effect
![Page 20: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/20.jpg)
A
C
B
D
X
Effect of X (ectopic expression)may be suppressed by l-o-f mutation in C
![Page 21: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/21.jpg)
7. Screen for modifier mutations
Screen for (EMS) mutations that enhance or suppress the ectopic expression phenotype
Map mutations by recombination or deficiency
Predict candidate genes from map location
Get mutants for candidate genes
Confirm genetic interactions
Similar relationship in human?
![Page 22: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/22.jpg)
Reverse genetics:From a gene to function
1. Candidate genes by location
Map positionFISHmapped genomic clonesgenome sequence
Known mutations mapped at same location
Molecular defect of the gene in mutantsSouthernexpression
![Page 23: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/23.jpg)
2. Gene knock-out by homologous recombination
3. Insertional mutaenesisscreen by PCR
4. Post-transcriptional inactivation Antisense RNA Ribozyme RNA interference (RNAi, dsRNA)
![Page 24: Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes](https://reader036.fdocuments.net/reader036/viewer/2022062407/56812a46550346895d8d80b1/html5/thumbnails/24.jpg)
5. Dominant-negative protein
e.g. Transcription factor
Domains: DNA-binding, Activation, Oligomerization
D- A+ O+ oligomer without DNA-binding
D+ A- O+ seq-specific repressor
D+ A- O- blocks target sites