Drosophila als Modell für die Analyse humaner

Drosophila als Modell für die Analyse
humaner Erkrankungen
Stefan Luschnig
[email protected]
Drosophila als Modell für die Analyse humaner Erkrankungen
Woche 1 (20.6. - 24.6.2016)
I: Genetik und Entwicklung von Drosophila melanogaster
Allgemeine Grundlagen: Genetik, Embryonal-Entwicklung von Drosophila
Tubuläre Morphogenese, Einfluss von Hypoxie auf die Entwicklung
Experimente:
1. Kartierung von Transposon-Insertionen (P-Elemente) durch inverse PCR und Sequenzanalyse
2. Analyse von Mutanten mit Defekten bei der tubulären Morphogenese: ImmunfluoreszenzFärbung, live imaging, konfokale Mikroskopie
Drosophila als Modell für die Analyse humaner Erkrankungen
Woche 1 (20.6. - 24.6.2016)
Drosophila as a model organism
Introduction
• Life cycle, development, genetics
Advantages:
• small, easy and cheap to culture
• large number of progeny (50 eggs per female per day)
• short generation time (9 days)
• rapid embryonic development (24h), accessible to live imaging approaches
• powerful genetic and genomic tools, cell lines
• extensive resources (collections of mutants, deletions, RNAi lines etc.) freely available
Disadvantages:
• labor-intensive maintenance of stocks (freezing not possible)
Life cycle
1 day
4 days
Generation time:
9 days (at 25°C)
1 day
1 day
2 days
Was können wir von Drosophila lernen?
1933 Thomas H. Morgan "for his discoveries concerning the role played by the
chromosome in heredity".
1946 Hermann J. Muller "for the discovery of the production of mutations by means
of X-ray irradiation".
1995 Edward Lewis, Christiane Nüsslein-Volhard and Eric Wieschaus "for their
discoveries concerning the genetic control of early embryonic development".
2011 one half jointly to Bruce Beutler and Jules Hoffmann "for their discoveries
concerning the activation of innate immunity" and the other half to Ralph Steinman
"for his discovery of the dendritic cell and its role in adaptive immunity".
Was können wir von Drosophila lernen?
• Genetik als Werkzeug für funktionelle Studien
Mutanten erlauben Einblicke in die Funktion eines
Genproduktes
Funktionsverlust
Phänotyp
Funktionsgewinn
Phänotyp
Faktor X
Loss-of-function
Gain-of-function
Mutanten erlauben Einblicke in die Funktion eines
Genproduktes
Funktionsverlust
Phänotyp
Funktionsgewinn
Phänotyp
Faktor X
Loss-of-function
Gain-of-function
Zell- und Entwicklungsdefekte
Mutanten erlauben Einblicke in die Funktion eines
Genproduktes
Funktionsverlust
Phänotyp
Funktionsgewinn
Phänotyp
Faktor X
Loss-of-function
Gain-of-function
in vivo Funktion
Zell- und Entwicklungsdefekte
Forward vs. reverse genetics
Forward genetics
phenotype > gene
•
Loss-of-function screens (e.g., for embryonic lethal mutations)
•
Gain-of-function screens (ectopic expression, overexpression)
•
Modifier screens (sensitized genetic backgrounds)
Reverse genetics
gene > phenotype
• targeted deletions (P-element excisions, Flp-FRT recombination)
• Gene targeting by homologous recombination (more recently: TALENs, CRISPR-Cas9)
type cells; tissue-specific transgenic RNAi in flies)
(in cultured
• RNAiwild
Drosophila genetics and genetic tools
•
•
•
Genome organization
•
•
•
•
•
Nomenclature
Balancer chromosomes
Mutagenesis (random vs. site-directed, mutagens, mapping strategies, genetic
screens)
Transposons
Transgenic animals
RNAi
Genetic mosaics
The Drosophila melanogaster genome
• 5 chromosomes (X,Y, II, III, IV): three autosome pairs, two sex chromosomes
• Genome sequenced (2000)
• 180 Mio bp (120 Mio bp Euchromatin)
• approximately 15.000 genes
• no meiotic recombination in males
40%
40%
Females: X/X; 2/2; 3/3; 4/4
Males:
X/Y; 2/2; 3/3; 4/4
http://flybase.org/
20%
Genomes of 12 Drosophila species
http://rana.lbl.gov/drosophila/
http://flybase.org/static_pages/species/sequenced_species.html
Genomes of 12 Drosophila species
Bhutkar et al. (2008) Genetics 179: 1657-1680.
complete genome sequences of 192 inbred
D. melanogaster lines
• complete genome sequences from
192 inbred lines derived from a
natural population are available
• This panel of inbred lines is being
used to study the genetics of
complex traits (life span, behavior,
etc.) > genome-wide association
studies
http://service004.hpc.ncsu.edu/mackay/Good_Mackay_site/DBRP.html
Polytene chromosomes
1000-2000 aligned stands of DNA; they form when successive rounds of
DNA replication are not followed by cell division (= endoreplication)
Somatic pairing
• Generally, homologous chromosomes align during prophase of meiosis I
• in few eukaryotes (diptera, some fungi), homologous chromosomes also align in somatic
cells, with important consequences for gene regulation and recombination
Polytene chromosomes: Cytology
X 1 -­‐ 20
2L 21 -­‐ 40
2R 41 -­‐ 60
3L 61 -­‐ 80
3R 81 -­‐ 100
4 101, 102
Examples:
white Sb 3B6 89B4-­‐6
Chromosomal deficiencies
Df(1)BA1
1A1; 2A
maintained
with Dup
1B2-14; 3A3
maintained
with Dup
3C2-3; 3E3-4
Df(1)N-8
T(1;3)sc[J4]
2F6; 3C5
Df(1)JC19
3C11; 3E4
Df(1)dm75e19
Df(1)A113
Df(1)JC70
4C15-16; 5A1-2
4F5; 5A13
Df(1)BA2-8
5A8-9; 5C5-6
Df(1)C149
Df(1)N73
6E2; 7A6
Df(1)Sxl-bt
7A2-3; 7C1
maintained
with Dup
7B2-4; 7C3-4
Df(1)ct4b1
Df(1)ct-J4
5C2; 5D5-6
3D6-E1; 4F5
maintained
with Dup
Df(1)dx81
5C3-10; 6C3-12
maintained
with Dup
7D1; 7D5-6
Df(1)C128
Df(1)RA2
7D10; 8A4-5
7F1-2; 8C6
Df(1)C52
Df(1)v-L15
Df(1)HA85
Df(1)N105
Df(1)C246
12A; 12E -or11F10; 12F1
12F5-6; 13A9-B1
maintained
with Dup
Df(1)sd72b
14B8; 14C1
14B13; 15A9; 35D-E
Tp(1;2)r[+]75c
Df(1)4b18
13F1; 14B1
13B5-6; 13E1-2
In(1)AC2[L]AB[R]
Df(1)RK4
Df(1)g
11D-E; 12A1-2
10F7; 11D1
10C1-2; 11A1-2
9F; 10C3-5
maintained
with Dup
Df(1)v-N48
9B1-2; 10A1-2
8E; 9C-D
Df(1)KA14
8B5-6; 8D8-9 -or8D1-2; 8E1-2
Df(1)lz-90b24
Df(1)RK2
Df(1)r-D1
12D2-E1; 13A2-5
Df(1)XR38
14A; 15D
maintained
with Dup
14C2-4; 15B2-C1
maintained
with Dup
Df(1)B25
15D3; 16A4-6
Df(1)BK10
15F2-9; 16C7-10
maintained
with Dup
18A5; 18D
Df(1)JA27
17A1; 18A2
Df(1)N19
16C; 16F
Df(1)RR79
Df(1)Exel6291
18A2; 18A3
18D13; 18F2
Df(1)HF396
18E1-2; 20
Df(1)A209
20A; 20F
Df(1)Exel6253
19F1-2; 20E-F
maintained
with Dup
Df(1)DCB1-35b
RpL36
RpL22
1
mRpL16
2
mRpL14
3
M(1)3E
mRpL33
mRpL30
4
RpL35
5
Rp7-8
RpL7A
6
RpL17
RpS14a
RpS14b
CG11386
RpS6
7
8
RpS28
9
Fs(1)10A
10
11
mRpL49
RpS15A
Hdl
CG12725
12
mRpL3a
mRpS25
RpL37
13
mRpL3
mRpS30
14
RpS19a
RpS19
mRpL22
15
RpS5
16
wupA
17
18
mRpS14
RpS10b
CG14224
19
CG15458
20
5
Df(2L)net-PMF
21A1:21B7-8
Df(2L)BSC16
21C3-4;21C6-8
Df(2L)dp-79b
22A2-3; 22D5-E1
41A-B; 42A2-3
41A
Df(2R)M41A4
Df(2R)nap9
Df(2R)ST1
43F; 44D3-8
46A; 46C
Df(2R)B5
45A6-7; 45E2-3
Df(2R)w45-30n
Df(2R)H3C1
42B3-5; 43E15-18
42A1-2; 42E6-F1
In(2R)bw[VDe2L]Cy[R]
42E; 44C
Df(2R)cn9
Df(2R)H3E1
44D1-4; 44F12
Df(2R)Np5
44F10; 45D9-E1
Df(2R)BSC29
45D3-4; 45F2-6
Df(2R)X1
46C; 47A1
Df(2R)stan1
47D3; 48B2
Df(2R)BSC3
50D1; 50D2-7
Df(2R)Exel7131
50E4; 50F6
Df(2R)BSC18
48E12-F4; 49A11-B6
Df(2R)en-A
46D7-9; 47F15-16
Df(2R)en30
48A3-4; 48C6-8
Df(2R)BSC39
48C5-D1; 48D5-E1
48E; 49A
Df(2R)CB21
Df(2R)vg-C
Df(2R)CX1
49C1-4; 50C23-D2
49A4-13; 49E7-F1
Df(2R)BSC40
March 2005
RpLP1
M(2)21AB
mRpL10
21
mRpL48
22
dpp
oho23B
23
24
RpL40
RpL27A
mRpL24
mRpS2
mRpL28
25
RpL37a
26
27
unmapped
female sterile
28
RpL36A
50D4; 50E4
Df(2R)Exel7130
Df(2R)BSC11
Df(2R)Jp1
51D3-8; 52F5-9
50E6-F1; 51E2-4
Df(2R)Jp8
52F5-9; 52F10-53A1
Df(2R)BSC49
Df(2R)robl-c
54B17-C4; 54C1-4
Df(2R)14H10Y-53
55A; 55F
Df(2R)PC4
Df(2R)BSC19
56F12-14; 57A4
56F11; 56F16
Df(2R)Exel7162
Df(2R)BSC22
56D7-E3; 56F9-12
56C4; 56D6-10
Df(2R)BSC26
55E2-4; 56C1-11
Df(2R)P34
54E5-7; 55B5-7
Df(2R)14H10W-35
54D1-2; 54E5-7
54C1-4; 54C1-4
54C1-4; 54C1-4
Df(2R)k10408
53D9-E1; 54B5-10
Df(2R)BSC45
Df(2R)AA21
Df(2R)Egfr5
57D2-8; 58D1
58D1; 59A
Df(2R)59AD
59A1-3; 59D1-4
Df(2R)X58-12
59B; 59D8-E1
Df(2R)ED4071
60F1; 60F5
Df(2R)Kr10
60C8; 60E7
Df(2R)Px2
60E6-8; 60F1-2
Df(2R)ES1
60E2-3; 60E11-12
Df(2R)M60E
60C5-6; 60D9-10
59D5-10; 60B3-8
Df(2R)or-BR6
Df(2R)vir130
56F9-17; 57D11-12
54C8-D1; 54E2-7
54B1-2; 54B7-10
Df(2R)BSC44
unmapped dominant
female sterile (most likely
corresponding to RpS13)
RpS13
29
mRpL51
30
sop
RpL13
RpL7
31
mRpS7
RpS27A
32
RpL9
33
CG5317
34
RpL24
mRpS23
35
mRpL4
36
RpS26
mRpL13
37
RpL30
mRpS18b
38
39
40
RpL21
RpL5
48E1-2; 48E2-10
Bloomington
Deficiency Kit
Df(2L)BSC4
21B7-C1;21C2-3
Df(2L)BSC106
21B8;21C4
Df(2L)ast2
21D1-2; 22B2-3
Df(2L)BSC37
22D2-3; 22F1-2
Df(2L)dpp[d14]
23C5-D1; 23E2
Df(2L)BSC28
23A1-2; 23C3-5
Df(2L)C14
22E4-F2; 22F3-23A1
Df(2L)JS17
23C1-2; 23E1-2
24C2-8;25C8-9
maintained
with Dup
Df(2L)Exel6011
Df(2L)E110
25F3-26A1; 26D3-11
25C8;25D5
Df(2L)sc19-8
24A2; 24D4
Df(2L)drm-P2
Df(2L)ed1 23F3-4;24A1-2
23E5; 23F4-5
Df(2L)BSC31
25C4;25C8
Df(2L)BSC109
Df(2L)cl-h3
25D2-4; 26B2-5
Df(2L)BSC5
Df(2L)BSC7
Df(2L)Dwee1-W05
27E2; 28D1
Df(2L)XE-3801
27C2-3; 27C4-5
26D10-E1; 27C1
26B1-2; 26D1-2
Df(2L)BSC6
26D3-E1; 26F4-7
27C1-2; 28A
Df(2L)spd[j2]
Df(2L)BSC4
Df(2L)TE29Aa-11
28E4-7; 29B2-C1
28A4-B1;28BD3-9
Df(2L)Trf-C6R31
28DE(within)
Df(2L)BSC53
Df(2L)BSC50
29C1-2; 30C8-9
Df(2L)N22-14
30C3-5; 30F1
Df(2L)J2
30F4-5; 31B1-4
32D1; 32D4-E1
Df(2L)Prl
Df(2L)BSC36
Df(2L)BSC32
32D1; 32F1-3
34A1; 34B7-9
Df(2L)r10
Df(2L)b87e25
Df(2L)TE35BC-24
35B4-6; 35F1-7
Df(2L)cact255rv64
35F-36A; 36D
Df(2L)C'
Deficiencies generated in the
Drosdel Project (isogenic
background)
Deficiencies generated by
Exelexis (isogenic background)
Deficiencies generated by the
Bloomington Stock Center using
the isogenic Exelexis collection of
FRT insertions
Deficiencies generated by the
Bloomington Stock Center using
the male recombination approach
40h35; 40h38L
40A5; 40D3
Df(2L)Exel6049
38A6-B1; 40A4-B1
Df(2L)TW161
37B2-12; 38D2-5
Df(2L)pr-A16
36C2-4; 37B9-C1
maintained
with Dup
Df(2L)TW137
35D1; 36A6-7
34B12-C1; 35B10-C1
Df(2L)BSC30
32F1-3; 33F1-2
Df(2L)FCK-20
32A1-2; 32C5-D1
31B; 32A
Df(2L)BSC17
29A2-B1; 29D2-E1
Definite Overlap Between
Deficiencies
Gap Minimized Between
Deficiencies
To Be Tested
Definite Gap
Known or Potential
Haplolethal or Haplosterile
Ribosomal Proteins
Other Ribosomal Proteins
Other Haplolethal or Haplosterile Loci
available for download at:
http://flystocks.bio.indiana.edu/df-kit-info.htm
RpL38
41
42
43
mRpL52
44
45
RpL31
46
mRpS32
CG1381
47
RpS15Ab
48
RpS11
49
mRpL18
50
mRpS16
RpS23
51
52
mRpL41
U
53
RpS15
RpPLP2
54
mRpS4
RpL18A
55
mRpS35
56
RpL11
RpS18
57
mRpL54
RpL29
58
mRpS29
RpS16
bonsai
RpS24
RpL23
59
RpL37b
RpL22-like
mRpL43
RpL12
RpL39
60
mRpS17
RpL41
RpL19
61A; 61D3
Df(3L)Exel6087
61A2; 62A7
Df(3L)emc-E12
61C5-8; 62A8
62A10-B1; 62D2
62F; 63D
maintained
with Dup
Df(3L)M21
Df(3L)Aprt-1
Df(3L)Ar14-8
Df(3L)R-G7
62B8-9; 62F2-5
Df(3L)BSC23
62E8; 63B5-6
63C2; 63F7
Df(3L)GN34
63E6-9; 64A8-9
Df(3L)HR119
Df(3L)GN24
63F4-7; 64C13-15
64C; 65C
65A2; 65E1
Df(3L)XDI98
65F3; 66B10
Df(3L)BSC13
Df(3L)Scf-R6
66E1-6; 66F1-6
66B12-C1; 66D2-4
Df(3L)pbl-X1
Df(3L)ZN47
Df(3L)BSC27
65D4-5; 65E4-6
Df(3L)BSC33
65E10-F1; 65F2-6
Df(3L)ZP1
66A17-20; 66C1-5
66B8-9; 66C9-10
Df(3L)66C-G28
Df(3L)h-i22
66D10-11; 66E1-2
Df(3L)BSC35
66F1-2; 67B2-3
Df(3L)AC1
67A2; 67D7-13 -or67A5; 67D9-13
Df(3L)BSC14
Df(3L)vin7
69F6-70A1; 70A1-2
Df(3L)BSC12
68C8-11; 69B4-5
67E3-7; 68A2-6
Df(3L)vin5
68A2-3; 69A1-3
69A4-5; 69D4-6
Df(3L)eyg[C1]
Df(3L)BSC10
69D4-5; 69F5-7
70A1-2; 70C3-4 +
small Df somewhere in 89
In(3LR)C190[L]Ubx[42TR]
70C1-2; 70D4-5
Df(3L)fz-GF3b
Df(3L)fz-M21
70D2-3; 71E4-5
Df(3L)XG-5
Df(3L)st-f13
74D3-75A1; 75B2-5
Df(3L)BSC8
72C1-D1; 73A3-4
71C2-3; 72B1-C1
Df(3L)brm11
71F1-4; 72D1-10
73A3; 74F
Df(3L)81k19
Df(3L)W10
75A6-7; 75C1-2
Df(3L)Cat
75B8; 75F1
75F2; 75F10
Df(3L)ri-XT1
77E2-4; 78A2-4
77A; 77D1
Df(3L)rdgC-co2
76B1-2; 76D5
Df(3L)kto2
Df(3L)ED4782
Df(3L)fz2
75F10-11; 76A1-5
76B4; 77B
Df(3L)XS-533
Df(3L)BSC20
76A7-B1; 76B4-5
Df(3L)ri-79c
77B-C; 77F-78A
77F3; 78C8-9
Df(3L)ME107
Df(3L)Pc-2q
78C5-6; 78E3-79A1
78D5; 79A1
Df(3L)ED4978
79C1-3; 79E3-8
Df(3L)Ten-m-AL29
Df(3L)HD1
79D3-E1; 79F3-6
79E5-F1; 80A2-3
Df(3L)BSC21
mRpL17
61
RpL23a
62
mRpL46
mRpL23
RpL8
mRpS28
63
RpL28
mRpS6
64
65
RpL18
mRpL50
66
mRpL36
RpL14
Df(3R)e1025-14
82F8-10; 83A1-3
Df(3R)ME15
81F3-6; 82F5-7
Df(3R)3-4
83A6; 83B6
Df(3R)Exel6144
82F3-4; 82F10-11
83B4-6
Df(3R)Tpl10 + Dp(3;3)Dfd[rv1]
(Bipartite Df)
83E1-2; 84A4-5
Df(3R)WIN11
83C1-2; 83D4-5
and 84A4-5; 84B1-2
Df(3R)ED5177
84A1; 84B1-2
Df(3R)Scr
Df(3R)BSC47
83B7-C1; 83C6-D1
Df(3R)Antp17
84B1-2; 84D11-12
-or- A6, D14
Df(3R)p712
84D4-6; 85B6
85A2; 85C1-2
Df(3R)p-XT103
Df(3R)BSC24
85C4-9; 85D12-14
Df(3R)by10
85F1-2; 86C7-8
Df(3R)BSC38
85D8-12; 85E7-F1
86C1; 87B1-5
Df(3R)M-Kx1
Df(3R)T-32
86E2-4; 87C6-7
Df(3R)ry615
87B11-13; 87E8-11
87D1-2; 88E5-6; Y
Tp(3;Y)ry506-85C
Df(3R)ea
88E7-13; 89A1
Df(3R)sbd105
Df(3R)P115
88F9-89A1; 89B9-10
89B9; 89C2-7
Df(3R)sbd104
89B7-8; 89E7-8; 20
maintained
with Dup
Df(3R)DG2
89E1-F4; 91B1-2
Df(3R)Cha7
91F1-2; 92D3-6
Df(3R)DI-BX12
Df(3R)BSC43
93B6-7; 93D2
Df(3R)e-R1
92F7-93A1;93B3-6
92B3; 92F13
Df(3R)H-B79
90F1-4; 91F5
93B; 94A3-8
Df(3R)23D1
94A3-4; 94D1-4
Df(3R)BSC55
95F7; 96A17-18
Df(3R)Exel6202
Df(3R)TI-P
96D1; 96E2
Df(3R)crb87-5
95A5-7; 95D6-11
Df(3R)mbc-R1
Df(3R)mbc-30
95A5-7; 95C10-11
94D2-10; 94E1-6
Df(3R)e-N19
Df(3R)BSC56
94E1-2; 94F1-2
95A4; 95B1
95C12; 95D8
95D7-11; 95F15
96DE2; 96E6
96F1; 97B1
97E3; 98A5
Df(3R)D605
97A; 98A1-2
Df(3R)Espl3
Df(3R)Exel6203
96A2-7; 96D2-4
maintained
with Dup
Df(3R)slo[8]
Df(3R)crb-F89-4
95D8; 95E5
Df(3R)Exel6197
Df(3R)Exel6196
95B1; 95D1
Df(3R)Exel9014
Df(3R)Exel6195
mRpL7-L12
RpS9
RpS17
67
mRpL2
68
RpL10Ab
69
mRpL20
RpS12
RpS4
70
NHP2
71
mRpL5
72
mRpS31
mRpS34
73
74
mRpS26
75
RpL26
76
mRpL21
mRpL15
77
78
RpLP0
79
80
Rp21
Qm
98B1-2; 98B3-5
Df(3R)BSC42
Df(3R)3450
98E3; 99A6-8
Df(3R)Dr-rv1
99A1-2; 99B6-11
Df(3R)L127
99B5-6; 99E4-F1
maintained
with Dup
Df(3R)B81
99C8; 100F5
maintained
with Dup
82
CG1172
RpL35A
mRpL44
83
RpL13A
Tpl
84
mRpS9
mRpL1
mRpS18c
mRpL19
85
RpL34b
mRpL47
RpS29
86
mRpL37
RpS25
RpL3
mRpL40
RpL24-like
87
mRpS21
88
mRpL11
RpS5b
RpL10Aa
mRpS10
mRpL9
89
mRpS33
Abd-B
CG16941
mRpS11
90
CG7215
91
mRpL55
92
RpS20
RpS30
93
mRpL35
94
mRpL45
RpS3
95
RpS19b
mRpS24
96
RpS27
RpL27
RpL34
97
RpS10a
98
mRpS22
RpL4
99
RpS8
RpS28a
RpL32
RpS7
mRpS18a
100
mRpL32
RpL6
Balancer chromosomes
Balancer chromosomes:
-­‐ carry mul@ple inversions to prevent meio@c recombina@on during female meiosis
-­‐ (usually) carry lethal muta@on(s) that lead to lethality of homozygous flies
Marker muta@ons:
-­‐ Dominant (or recessive) muta@ons with a visible phenotype: Balancer chromosomes
do balancer chromosomes really prevent recombina@on? Drosophila mutations
Drosophila genetic nomenclature
Gene named aRer first mutant phenotype (not necessarily the null mutant phenotype); examples:
-­‐ white : lacks all eye pigment
-­‐ apterous , wingless : lack wings
-­‐ eyeless : lacks eyes
One of the biggest classes of visible mutants affects eye pigmenta@on (>80); e.g.:
brown, carmine, carna7on, cinnabar, claret, deep orange, garnet, karmoisin, light, lightoid, orange, pink, purple, purploid, ruby, scarlet, vermilion, white
Some very inven@ve names; e.g.:
-­‐ ken and barbie, technical knockout, Cubitus interruptus, saxophone
Mutagens
EMS (Ethyl methane sulphonate)
-­‐ most efficient chemical mutagen (best compromise between toxicity and mutagenicity)
-­‐ highly reac@ve alkyla@ng agent, causes mostly GC -­‐> AT transi@ons
-­‐ delivered by feeding male flies
-­‐ cause many muta@ons throughout the genome
-­‐ mapping is not trivial and work-­‐intensive (“needle in a haystack”)
X-­‐rays
-­‐ cause chromosomal breaks, which, when repaired result in dele@ons, duplica@ons, inversions
-­‐ less efficient than chemical mutagens
-­‐ cause cytologically visible defects, oRen complex genomic rearrangements
Transposons (P elements, PiggyBac elements etc. )
-­‐ inser@onal mutagenesis
-­‐ rapid iden@fica@on of affected gene
-­‐ low efficiency
-­‐ inser@on sites not en@rely random (P-­‐elements: oRen in introns, 5´-­‐UTRs)
-­‐ inser@on sites are compara@vely easy to map Transposons
P element:
naturally occurring transposon
requirements for transposi@on:
-­‐ P element ends in cis
-­‐ P transposase in trans
+/-­‐ random inser@on; preference for promoter regions
main tool for crea@ng transgenic flies
inser@ons can also disrupt gene func@on
Transgenesis
Transposase mobilisiert rekombinantes P-Element auf
zweitem Plasmid
Integration erfolgt an zufälliger Stelle im Genom
Venken, K. J. T. et al., Development 2007; 134:3571-3584
Transgenesis
Mosaik
Problems:
integra@on site random
posi@on effects influence expression of transgenes
Venken, K. J. T. et al., Development 2007; 134:3571-3584
Mapping of P element (or other
transposon) insertions
•
genetic mapping: Segregation analysis (> mapping to a
chromosome), recombination mapping (> mapping to a
region on a chromosome)
•
cytological mapping: in situ hybridization on polytene
chromosomes
•
molecular mapping: inverse PCR (or similarly: plasmid
rescue)
Inverse PCR (iPCR)
Inverse PCR (iPCR)
•
Sie erhalten zwei Fliegenstämme, die jeweils ein P-Element-Transgen (pUASTKonstrukte) an unbekannter Stelle im Genom integriert haben.
•
Ihre Aufgabe ist es die Insertionsstellen dieser Elemente mit Hilfe der inversen PCR
Basen-genau zu bestimmen.
•
•
•
•
•
Zeitplan (siehe Skript):
•
Woche 2: Analyse von Sequenzen, BLAST
Montag: Isolation von genomischer DNA aus Fliegen
Dienstag: Restriktionsverdau (über Mittag), Ligation (über Nacht)
Mittwoch: Fällung der ligierten DNA, PCR
Donnerstag: Agarose-Gelelektrophorese,Vorbereitung der Proben zum
Sequenzieren, Sequenzierung (außer Haus)
Transgenesis:
The ΦC31 integration system
agP + agB -­‐> integrase -­‐> agL + agR
Transgenesis:
The ΦC31 integration system
Bischof J. et al., PNAS 2007; 104: 3312-3317
The genetic toolbox
• polytene chromosomes
• many mutations available
• balancer chromosomes
• molecularly defined deletions and duplications covering the genome
• transposon-mediated germline transformation
• overexpression, ectopic expression, conditional expression: Gal4/UAS system
• transgenic RNAi (tissue-specific knock-down of gene function)
• CRISPR/Cas9-based genome editing
• clonal analysis (genetic mosaics)
• tissue culture (S2 cells; genome-wide RNAi screens)
• databases! FlyBase
The GAL4-UAS system
Gal4 ‘driver’ line
UAS ‘responder’ line
(UAS promoter is
silent in the absence
of GAL4 -> no GFP
expression)
(Yeast GAL4 protein
expressed under the
control of a Drosophila
promoter)
breathless-Gal4 UAS-GFP
GAL4 activates transcription of UAS
construct -> GFP expression (only in
cells that express GAL4):
The GAL4-UAS system
Modular system:
• Large number of Gal4 and UAS lines available
• GAL4 lines for various developmental stages, cell types, organs, inducible
versions of GAL4
• UAS-gene-of-interest, fluorescent proteins, toxins, RNAi (dsRNA), CRISPR
(sgRNA) constructs...
• Useful for overexpression, ectopic (misexpression), and inducible expression of
genes, as well as for knocking down gene expression (RNAi)
RNA interference
Mohr et al. Nature Reviews Molecular Cell Biology15, 591–600 (2014)
tissue-specific transgenic RNAi
Dietzl et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156.
genome-wide RNAi screens
in vivo
in cultured cells
Ron Vale lab, UCSF
Mummery-Widmer et al. (2009) Nature 458, 987–992.
problems with RNAi
• efficiency of knock-down (false-negative results)
• specificity of knockdown (off-target effects)
How to validate results of RNAi experiments?
Mohr et al. Nature Reviews Molecular Cell Biology15, 591–600 (2014)
Genetische Mosaike
Organismus mit Zellen verschiedener Genotypen, die aus einer Zygote hervorgegangen sind
(<> Chimäre: unterschiedlicher zygotischer Ursprung)
Möglichkeiten der Entstehung von Mosaiken:
- Verlust von Chromosomen während der Entwicklung (instabile Chromosomen, z.B. Ring-X
Chromosom)
Bsp. Gynandromorphe bei Insekten: aus männlichen und weiblichen Zellen mosaikartig
zusammengesetzte Individuen
- somatische Mutationen
- mitotische Rekombination
mitotische Rekombination
>
chromosome segregation
*
>
*
>
>
heterozygous cells
St Johnston D., Nature Reviews Genetics 2002; 3: 176-188
Anwendungen von genetischen Mosaiken
• Zell-Schicksalskarten (”fate maps”):
zu welchen Strukturen kann sich eine
Vorläuferzellen im adulten Tier entwickeln?
wie gross kann ein Klon maximal werden?
Kompartiment-Grenzen
> Analyse von Klonen markierter Zellen, z.B. mit
sichtbaren Mutationen (mwh)
mwh/mwh
multiple wing hairs (mwh)
mwh/+
Anwendungen von genetischen Mosaiken
• klonale Analyse zum Bestimmen der Zell-Autonomie von Mutationen (Bsp. SignalTransduktionswege): Hat das Ausschalten eines Gens in einer Zelle Auswirkungen
auf benachbarte wildtypische Zellen, oder nur auf die mutante Zelle selbst ?
Zell-autonome Mutationen wirken sich in genetischen Mosaiken nur auf die mutante
Zelle, jedoch nicht auf benachbarte wildtypische Zellen aus (z.B. Rezeptor)
Nicht-Zell-autonome Mutationen wirken auf entfernt liegende Zellen (z.B. diffusible
Signalmoleküle, Hormone)
Anwendungen von genetischen Mosaiken
• Analyse von homozygoten Klonen letaler Mutationen im heterozygoten Organismus
(z.B. Analyse der Funktion von essentiellen Genen während der Oogenese:
Keimbahnklone)
• > Genetische screens in Mosaik-Tieren
Types of mutant alleles: Muller´s morphs
scale of normal gene (allele) function / activity
100%
0%
loss of function
Antimorph
Counteracts
wild-type allele
(“dominantnegative”)
Amorph
Complete loss
of function
(“null”)
Hypomorph
Reduced
normal
function
dominant
recessive
mutation in a
protein that
acts as a dimer,
such that the
dimer still
forms, but is
inactive (e.g.,
Receptor
tyrosine kinase)
deletion of
a gene
gain of function
Hypermorph
Increased
normal
function
Neomorph
Qualitatively
new /different
function
recessive
dominant
dominant
mis-sense mutation
in a protein that
leads to reduced
activity
point mutation that leads
to constitutive.g., missense mutation in a
proteine activity of a
protein (kinase, G-protein)
regulatory
mutation that
leads to ectopic
expression of a
protein
Wild type
Normal
function
https://en.wikipedia.org/wiki/Hermann_Joseph_Muller
Genetic tools and screens I
•
classic screens for genes controlling embryonic patterning:
the ‘Heidelberg screen’
•
•
maternal vs. zygotic genes
•
strengths and limitations of classic loss-of-function screens
genetic tools for morphological screens with specific
readouts
Christiane Nüsslein-Volhard and Eric Wieschaus
• 1978/79: set out to identify all genes
required for embryonic patterning using a
random mutagenesis approach
• First large-scale systematic genetic screen
in Drosophila
• Nobel Prize for Medicine or Physiology
1995
• basic idea: randomly mutate all genes and
identify those required for embryonic
patterning by their specific phenotypes
• requires a morphological readout for
embryonic pattern
The larval cuticle provides a readout
of positional information
imaginal discs
brain
dorsal epidermis
germline
posterior midgut
mesoderm
ventral epidermis
head
anterior midgut
dorsal epidermis
ventral epidermis
Head Thorax
•
•
Abdomen
Telson
cuticle: positional information, polarity, differentiation
easy and quick to prepare, scalable procedure
zygotic vs. maternal mutations
zygotic mutations
maternal-effect mutations
mutagenesis screens
mutagens:
•
•
•
chemical (e.g., alkylating agents: EMS, ENU; epoxides)
physical (e.g., radiation: X-rays)
biological (transposons: P-element, PiggyBac, Minos, Mariner, Hobo, etc.)
•
EMS (Ethylmethane-sulfonate) causes mainly (not exclusively) G to A
transitions; best compromise between mutagenic efficiency, toxicity, and
ease of handling
Crossing scheme for mutagenesis of the 2nd
chromosome (F3 screen)
• cn bw: isogenized chromosome with
recessive markers
25mM EMS
DTS
cn bw
x
CyO
cn bw
• 25 mM EMS generates on average
0.6 lethal mutations per chromosome
* cn bw
CyO
F1
DTS
x
or
* cn bw
• DTS: dominant temperature-sensitive
lethal mutation
CyO
• CyO: balancer chromosome
(homozygous lethal)
DTS
14‘000
single malecrosses
F2
* cn bw
CyO
grow at 29°C
x
* cn bw
* cn bw
DTS
DTS
CyO
CyO
DTS
CyO
DTS
CyO
Test each line for the presence of 25% non-hatched embryos
Prepare cuticles and analyze phenotypes
F3
* cn bw
* cn bw
* cn bw
CyO
* cn bw
CyO
CyO
CyO
25% homozygous embryos
Viable F3 lines were screened for maternaleffect mutations
• F3 flies, if homozygous viable, can
be screened for female fertility
F3
* cn bw
* cn bw
* cn bw
CyO
* cn bw
CyO
CyO
CyO
test homozygous viable F3 females
for maternal-effect mutations
viable progeny or 100% mutant embryos?
• Strict maternal effect:
gene product is not required for
zygotic development
• 100% of the progeny of maternaleffect mutants show a phenotype
• heterozygous (balanced) F3 flies are
used maintain the mutant stock
Results of the Heidelberg screen (all
chromosomes)
• 27’000 lines tested:
– 18’000 lethal hits
– 25 % (4’300) are embryonic-lethal
– 13% (586) of these show embryonic visible (i.e. patterning) phenotypes
– on average 5-6 alleles per complementation group
• 140 genes with specific patterning defects, 30 of these are strictly maternally
required genes
• Expression patterns of cloned genes correspond to mutant phenotype
• Basis for current understanding of early embryonic development
Cuticle phenotypes of embryonic lethal mutants
Jürgens et al. (1984), Roux’s Arch Dev Biol 193, 283
three classes of zygotic genes control
segmentation
gap
pair rule
segment
polarity
Three classes of maternal genes
define the anterior-posterior axis
Saturation mutagenesis
•
•
How many genes were hit and how many were missed?
how to estimate the degree of saturation?
rate of discovery of new loci:
Jürgens et al. 1984
distribution of allele frequencies
(Poisson distribution):
Nüsslein-Volhard et al. 1984
summary: chemical mutagenesis screens
• Strengths:
– Unbiased approach
– In principle, all genes can be mutagenized (but not all genes are equal)
– Different types of mutations are generated: null, hypomorphic, dominantnegative, gain-of-function (allelic series)
• Limitations:
– Mutation frequency is gene-specific and can vary to a large degree. Small
genes are less frequently hit (e.g. miRNA genes; ‘hot spots’ vs. ‘cold spots’)
– F2 & F3 type screens are very labor-intensive (establishment and
maintenance of a large number of stocks)
– Genes with redundant functions escape detection
– mapping / identification of mutated genes is time-consuming (rate-limiting
step; new sequencing techniques accelerate mutation detection)
Limitations of loss-of-function screens
Mutations that could not be identified:
maternal-effect genes
genes involved in patterning of internal structures
redundant genes
Enormous workload (individual crosses !)
Only the first essential function of a gene can be analyzed
Analysis cannot be limited to a particular tissue
Identification of mutations is tedious
Dominant modifier screens
Idea:
Render recessive mutations dominant (haplo-insufficient)
Strategy:
Generate a “sensitized” situation in which 50% of normal gene
product is no longer sufficient
-> F1 dominant modifier screens
The compound eye of Drosophila as a
model organ for genetic screens
The Compound Eye –
A Model System for Cell Signaling
3
2
1
4
7
5
6
Each
ommatidium
contains
eight
Jedes Ommatidium besteht aus
photoreceptor
cells(R1-R8)
(R1-R8)
8 Photoreptorzellen
Trapezartige Anordn. der Rhabdomere
R-Zellen
mit charakteristischse
Struktur
(Mikrovillisaum
– Rhabdomer mit
lichabsorbierenden
Rhabdomere:
charakteristische Struktur,
Mikrovilli-Saum
mit lichtabsorbierenden
Pigment
(Rhodopsin)
Entwicklung der Ommatidien
Augen-Imaginalscheibe Differenzierung von posterior
nach anterior
Bride of sevenless (Boss) kodiert für ein
Signalmolekül, Sevenless für den Rezeptor
zusätzliche R7Zellen
zu viel oder
ektopisches
Boss Signal,
oder konstitutiv
aktiver Sev
Rezeptor
boss wirkt nicht-zellautonom
sevenless Allele mit gegensätzlichen Phänotypen:
Funktionsverlust vs. Funktionsgewinn
Wildtyp
sevenless loss-offunction:
R7-Zellen fehlen
sevenless gain-offunction: zusätzliche
R7-Zellen
A dominant suppressor of the S11
activated
Dominant suppressors of Sev
Sevenless receptor
SevS11; +/+
SevS11; dos/+
mutation of a single copy of the daughter of sevenless (dos) gene dominantly suppresses
the rough eye phenotype caused by activated Sevenless
Der Ras/MAPK
Signaltransduktionsweg
Die Sevenless Signalkette
Transkriptionsaktivatoren
Pointed (Pnt)
Seven in absentia (Sina)
Transkriptionsrepressor
Yan
Dominant modifier screens
Advantages
- F1 screen: rapid, efficient
- High degree of saturation reachable
- Genes with an early essential function can be detected
- Sets of molecularly defined deletions or UAS-RNAi lines can be used to
systematically sample the genome
Disadvantages
- 50% reduction in gene expression is often not sufficient
-> special (dominant-negative) alleles
- Efficiency is pathway specific
- Identification of mutations can be tedious
Mosaic screens
Idea:
Generate homozygous mutant tissue in the F1 generation
-> F1 FLP/FRT screen for recessive mutations
- random
- tissue specific
The ey-FLP/FRT system
Certain tissues are made homozygous mutant using the FRT
system and a tissue specific FLP
-> recessive mutations can be screened in F1
St Johnston D., Nature Reviews Genetics 2002; 3: 176-188
Changes in insulin signaling activity result in
variable head sizes
chico
pinhead
control
PTEN
bighead
The “pinhead” screen
300‘000 flies screened
580 small head
mutations
~ 30 growth-promoting
genes
137 big head
mutations
17 growth-inhibiting
genes
The pinhead screen revealed components
of the insulin and TOR signaling pathways
Insulin
PIP2
Chico
InR
PIP3
PTEN
PH
PI3K
PH
PKB
PDK1
Tsc2 Tsc1
Raptor
TOR
S6K
4EBP1
FLP/FRT mosaic screens
Advantages
- F1 screen !
- High degree of saturation reachable
- Allelic series (alleles of varying strength)
- Genes with an early essential function can be detected
Disadvantages
- Genes with redundant functions cannot be detected
- Genes with weak phenotypes escape detection
- Only genes with cell/tissue autonomous functions can be detected
- Identification of mutations is tedious
Principle of over-expression screens
GMR
Gal4
UAS
EP
EPElement
element
GMR-Gal4 + UAS-InR
endogenes
endogenousGen
geneXX
X
Tester strain
GMR-Gal4
UAS-InR
EP lines
Suppression of big eye phenotype?
Overexpression of Imp-L2 suppresses
InR-mediated overgrowth
GMR-Gal4 UAS-InR
GMR-Gal4 UAS-InR
+ EP 5.66
GMR-Gal4 UAS-InR
+ EP 5.66mut
Imp-L2: Imaginal morphogenesis protein-Late 2; encodes a secreted insulin antagonist
Hugo Stocker, ETH Zurich
Imp-L2 overexpression reduces growth