Download 模式动物果蝇 在生命科学和医学研究中的应用

利用模式动物果蝇
研究神经发育和神经疾病
概述: 1，果蝇在生命科学研究中的历史贡献
2，来源于果蝇研究中的重大技术突破
3，果蝇在现代医学研究中的应用
专题: 以果蝇为模型研究重大神经疾病的分子遗传机制
Why work with flies?
•Short life cycle (~10 days)
•Highly prolific (>1000 offsprings / female)
•Economical
•Harmless to humans and environment
•Century long accumulation of genetic tools
Practically, flies are an excellent
model system
Drosophila has a minimum set of chromosomes
成虫
4d
胚胎
1d
蛹
10hrs
1.5d
1d
2d
幼虫
~10 days at 250C
Most Commonly Used Mutants
wildtype, WT
white
yellow
Curly
Bar
ebony
Basic Genetics Concepts
Mutants--lots and lots of various mutants
Mutations--loss-of-function and gain-of-function mutations
Mutagen--chemical, physical and biological mutagens
Mutagenesis--generation of various mutants
Mosaics--mutant tissues or cells in otherwise wildtype individual
Epistasis--gene interaction in terms of phenotypes
Genotype versus phenotype
Forward genetics versus Reverse Genetics
Genetics Versus Biochemistry
Biochemistry: the scientific study of the chemistry of living cells,
tissues, organs and organisms; in vitro structural and functional
study of purified living components
Genetics: the study of the patterns of inheritance of specific traits;
understanding the in vivo functions of a gene through mutants
Interdisciplinary approach
to study biological processes
Protein: 蛋白、蛋白质
Mutation/mutants: 突变、突变体
The Nobel Prize in Physiology or Medicine 1933
For his discoveries concerning the role played by the chromosome in heredity
Morgan, T.H. Science 32: 120-122 (1910) Sex limited inheritance in Drosophila
From Columbia University to California Institute of Technology
Morgan and His Three Extremely Gifted Students
Alfred H. Sturtevant
Calvin B. Bridges
Hermann J. Muller
A. H. Sturtevant
The Nobel prize in Physiology or Medicine, 1946
For the discover of the production of mutations by means of X-ray irradiation
Hermann Joseph Muller, 1890-1967
Indiana University, Bloomington, IN, USA
Before his discovery, mutants were largely from spontaneous mutations!!
It revolutionized animal and plant breeding, offered novel insights
into irradiation damage and cancer biology
Ed B. Lewis, 1918-2004
Nobel Prize Winner in Physiology or Medicine, 1995
Lewis, E.B. (1978) A Gene Complex Controlling Segmentation in Drosophila. Nature 276, 565-570
Homeotic gene expression--colinearity
All transcription factors
The Nobel Prize in Physiology or Medicine, 1995
For their discoveries concerning the genetic control of early embryonic development
Edward B. Lewis
77
Christiane Nusslein-Volhard
52
Eric F. Wieschaus
48
Nuesslein-Volhard, C., Wieschaus, E. (1980).
Mutations Affecting Segment Number and Polarity in Drosophila. Nature 287, 795-801
Some of these genes may be responsible for spontaneous abortions and
congenital malformations
Walter Gehring, University of Basel, Switzerland
Those who succeed…
EMBL, Heidelberg
…are the most hard-working and persistent.
A Turning Point in the History
of Developmental Genetics
The October 30, 1980 cover
“Mutations Affecting Segment Number and Polarity in Drosophila”
Classical F3 Screen for Recessive Mutations
EMS
propagation
Self-cross
Balanced homozygotes:
Dia as larvae
Balanced mutant stock:
viable
Homozygous for mutations:
Screen embryos for phenotypes
Tedious and time-consuming: single crosses, three generations
Mutations and Genes Identified from the Screen
WT
wingless
staufen
150 development-regulating genes that affect gross morphology in
Drosophila
stimulated the search for development genes in other systems
(nematode and mouse)
virtually all the genes involved in development of Drosophila are
represented also in vertebrates, conserved over 700 million years of
evolution
The Uniqueness of the Nobel Prize Screen
1, Screen for phenotypes in embryo instead of adult (1965-1980)
2, the first screen to find mutations affecting a given process
Developmental features crucial for the success of the screen
Larval cuticle: an excellent readout of embryo patterning
Embryogenesis is very fast, housekeeping proteins are
provided from the egg. Only the patterning genes are
specifically identified.
The Nobel Prize in Physiology or Medicine, 1995
For their discoveries concerning the genetic control of early embryonic development
Edward B. Lewis
Christiane Nusslein-Volhard
Eric F. Wieschaus
Determined to understand a biological process and be persistent!
We were young and foolish, and it was worth trying
Thomas Hunt Morgan Medal
Genetics Society of America MedalGeorge W. Beadle Medal
1983
1995
1958
7 Nobel Prize Winners
2003
1995
2003
2003
Thomas Hunt Morgan Medal
Genetics Society of America MedalGeorge W. Beadle Medal
12 recipients
Michael Ashburner
Gene regulation: chromosomal puffs
Books: most popular books
Genome: successful fly genome projects
Database: most comprehensive flybase
Department of Genetics
Cambridge University
David S. Hogness: The 2003 Thomas Hunt Morgan Medal
Department of Biochemistry, Stanford University
David Hogness and son, Peter
David S. Hogness: The 2003 Thomas Hunt Morgan Medal
Department of Biochemistry, Stanford University
application of mol. biol. to fly genetics
1, produced first genomic library of any organism
2, Established the method of “positional cloning” or “map-based cloning”
3, Establsihed “colony hybridization”, 1975
4, First person to practice “genomics” and “functional genomics”
5, Discovered “Goldberg-Hogness box”, now known as the TATA box,
6, Trained many well-known scientists, eg., Gerry Rubin, Richard Mann
Two Most Active Players in the Fly Community in the Last Twenty Years
Gerry M. Rubin
UC Berkeley
Vice President of the HHMI
Allan C. Spradling, Director of
the Department of Embryology of
the Carnegie Institute
Rubin, G.M., Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors.
Science, 1982 218(4570):348—353
Prominent leaders in the Drosophila Genome Project
Gerry: the Berkeley Drosophila Genome Project, sequencing of genomic clones,
EST clones and mapping of P element insertions. The genome sequence of Drosophila
melanogaster. Science 2000 287(5461):2185--2195
Allan: Genetic screens to mutate the genome by P elements, aiming at having a P
insertion in each of the 15,000 genes. The BDGP gene disruption project: single
transposon insertions associated with 40% of Drosophila genes.
模式动物果蝇
在生命科学和医学研究中的应用
1，果蝇在生命科学研究中的历史贡献
2，来源于果蝇研究中的重大技术突破
(P Insertion; UAS-GAL4; FRT-FLP)
3，果蝇在现代医学研究中的应用
Landmarks in Fly Genetics
• Elucidation of early development by systematic
genetic screens, 1980; Nobel Prize in 1995
• Germline transformation of fruit flies, 1983
• Completion of Genome Sequencing in 2000, fly as
a model for human diseases
• Mutagenesis by P element insertion, each gene has
a insertion
• UAS-GAL4 system to manipulate gene expression
• FRT-FLP system to make mosaic clones
Gene Expression Manipulation
UAS-GAL4 System Derived From Yeast
Brand, A.H., Perrimon, N.
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.
DEVELOPMENT 1993 118(2):401--415
Rorth, P.
A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. PNAS 1996
93(22):12418--12422
Rorth, P., Szabo, K., Bailey, A., Laverty, T., Rehm, J., Rubin, G., Weigmann, K., Milan, M., Benes,
V., Ansorge, W., Cohen, S.
Systematic gain-of-function genetics in Drosophila. DEVELOPMENT 1998 125(6):1049--1057
GAL4–UAS System for Directed Gene Expression
GAL4: a transcription factor
UAS: upstream activating sequence
Chromosomal Recomb. —Mosaic Analyses
FRT: a specific DNA sequence
FRT-FLP System
FLP: flippase or FLP recombinase
1, Golic KG, Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination
in the Drosophila genome. CELL 1989 59:499--509
2, Golic, K.G. Site-specific recombination between homologous chromosomes in
Drosophila. SCIENCE 1991 252:958--961
3, Xu, T., Rubin, G.M. Analysis of genetic mosaics in developing and adult Drosophila tissues.
DEVELOPMENT 1993 117(4):1223--1237
4, Lee, T., Luo, L. Mosaic analysis with a repressible neurotechnique cell marker for studies of
gene function in neuronal morphogenesis. NEURON 1999 22(3):451--461
Mosaic Analyses—Powerful Genetic Tool
conditional knockout
Parental Cell
DNAReplication
duplication
DNA
Indu. Recombination
Daughter
cells
GFP+ clone
A
X
X
X
X
X
No recombination
B
>
>
偷梁换柱
X
GFP supp
>
X
>
X
>
X
>
X
>
GFP supp
>
GFP supp
>
GFP supp
>
GFP supp
Recombination induced by yeast derived Flipase and FRT sequences
Advanced Mosaic Analysis--MARCM
Lee T & Luo L, Neuron, 1999
Mutant, no label
classical
WT, labeled
Mutant, labeled
MARCM
WT, no label
Single Brain Neuron
When FMRP Expression is Altered
WT
Mutant
Overexpression
模式动物果蝇
在生命科学和医学研究中的应用
1，果蝇在生命科学研究中的历史贡献
2，果蝇研究中的重大技术突破
3，果蝇在现代医学研究中的应用
果蝇在医学研究中的应用
1, Infection and immunology
2, Cancer biology and signal transduction
3, Neurobiology: ion channels, circadian rhythm, learning and memory
4, Neurological diseases: neurodegenerative disease and mental retardation
A Good Model to Study Cancer Biology
A Good Model to Study Cancer Biology
Fly Models of Neurodegenerative Diseases
Drosophila Eye is an Ideal System to Study Neurodegeneration
小结
Fly Community as a Close Family
• You have to come to a fly lab to learn fly genetics.
We have a well kept fly researcher phylogeny
• The best database for a model organism:
http://flybase.bio.indiana.edu
• A well maintained stock center and genomic
resources center
• Easy and willing to share reagents privately:
stocks, antibodies, constructs etc.
• A fly community is established in China!
Fly Albums