Transcript Drosophila melanogaster

Drosophila Genetic Laboratory
PI: 廖國楨/孫以瀚
The Basics of Fly Genetics:
Information
a. Books




Drosophila: A laboratory handbook
The genome of Drosophila melanogaster
The development of Drosophila melanogaster
The embryonic development of Drosophila
melanogaster
 Drosophila: A practical approach
 Fly pushing
b. The Flybase http://flybase.bio.indiana.edu
mirror site http://flybase.nhri.org.tw
Drosophila stocks
1. Wild-type stocks: Oregon-R and Canton-S
2. Mutant Stocks: Bloomington and Umea Stock Centers
Fly Chromosomes
Female
X
2L
2R
3L
Male
Y
 Genetic map: e.g. 2-57 cM (centi-Morgan)
 No recombination in males
3R
4
Type of Mutations
• Point mutation
• Inversion
(
)
• Translocation
• Compound chromosome
• Duplication
• Deficiency
( )
Polytene chromosome
Physical mapping for
chromosomal rearrangement,
duplication and deficiency.
X: 1-20
2L: 21-40
2R: 41-60
3L: 61-80
3R: 81-100
4: 101-104
Fly Morphology
Notum
Recognized Markers are the key
to decipher genotypes. Mutations
affecting body (color), eye (color
and shape), wing
(shape and vein) and bristle
(shape and color)
Examples:
Eye shape and color
wild type
Bristle
Bar
Sco
Body color
Bristle shape
Sb and Hu
Wing shape
wild type
CyO
Nomenclature
f; cn bw/CyO; TM3 Sb1 e1/ttkosn
Rules
•
order: X/Y; 2nd; 3rd; 4th chromosome
•
genotype: italicized
•
mutant names are abbreviated with three letters or fewer
•
TM stands for third multiple - a balancer chromosome
•
low case indicate recessive phenotype
•
uppercase in the first position indicate dominant phenotype
•
semi-colon separate genotype symbols
•
a genotype written on a single represent mutation on that
chromosome is homozygous
•
anything that is not shown is presumed to be wild type;
similarly, heterozygosity is only indicated by the mutant loci
•
allelic name is shown by superscript
•
eyD indicates that this particular allele is dominant. The gene
symbols represent that original allele is recessive.
FM: first multiple
SM: second multiple
TM: third multiple
Balancer Chromosomes
Features:
• Homozygous lethal except FM
• Multiple inversion
inhibit recombination
• At least one dominant marker
function to trace
chromosome and to
maintain stock
Mating Scheme
FM7a ; CyO
FM7a Sco
X
virgin
females
nd
nd
or
Y
FM7a ; CyO
nd
+
Culture Condition
25 ℃ and 60% humidity
multiple
Basic Fly Husbandry
Working with Drosophila doesn’t require much technical skill.
Care is essential.
The power of fly genetics comes from the ability to perform
cross in which each possible genotype in the progeny is
recognized easily and unambiguously. Virgin females used
in crosses are important factor. If flies are not healthy, it is
important to recover enough of the progeny needed to
continue the experiment. Rejuvenilization may be help.
The healthiest way to keep fly stocks is at 25 ℃, 60%
relative humidity. Temperature ranges from 29 ℃ to 18 ℃.
True breeding stocks can be transferred w/o anesthetization.
Method: Tap → off plug → new vial → tap flies to new vial →
put plug back on.
Usually, 10-15 females per vial are sufficient.
Coddling difficult strains: Use a fresh food vial garnished with
fresh yeast paste (baker’s dried active yeast mixed to the
consistency of peanut butter), keep the culture in ideal
conditions (25 ℃, 60% RH), and say an occasional prayer.
Foods:
1. Flour-treacle-agar
2. Yeast-glucose-agar
3. Instant potato medium
4. Casein-based defined medium
5. Yeast-cornmeal-molasses-agar
6. Apple juice medium
Pests
1. Fungal control – Tegosept
2. Bacterial control – penicillin G (less problem)
3. Mite control -
For new stocks:
(a) Keep all incoming stocks in quarantine
(b) Make sure they are mite-free before put them into your fly room
(c) The stocks quarantine for at least two generations
Good housekeeping will help to reduce happening of mite infection:
1. Autoclave all infected bottles and vials
2. Clean your bench, microscope and fly handle equipments regularly
3. Remove all old vials from fly room
4. Use cotton plug instead of foam plug
5. Use Tedion when mite infection becomes a serious problem
Tedion (2,4,5,4’-tetrochloro-diphenyl sulfone) is
dissolved in acetone at the final conc. 5000 ppm.
Soak filter paper and leave them dry in the hood.
Benzyl benzote can also be used in mite control. Make
50% solution in ethanol and soak filter paper. This
chemical may kill some stocks.
Anaesthetizers:
1. Ether
(a) Tip the flies to the bottom of
vial or bottle.
(b) Remove the plug and rapidly
invert over the mouth of
etherizer.
(c) Replug the vial or bottle.
(d) Wait until all flies have
stopped moving.
(e) Tip them on to the glass plate
for examination.
Two etherizers and one
apparatus to prevent food from
falling
2. Carbon dioxide
(a) Tip flies into the
anaesthetizer.
(b) Transfer to the porous
ethylene film with CO2
the flowing through.
Replace by a needle
An apparatus using CO2 fir anaesthetizing flies
Carbon dioxide has a number of advantages:
(a) It is safer and more pleasant for the user
(b) Flies are anaesthetized more quickly
(c) They recover consciousness rapidly and will mate whereas
ether delays mating
3. Triethylamine: usage is the same as ether. The anaesthetized effect
is much longer than ether, but its toxicity is also higher.
Collecting Flies for Crosses
** All genetic crosses, females have to be virgin **
Virgins can be distinguished by their pale pigmentation, by their
morphology, or by the presence of a dark spot in their abdomens .
Alternatively, they can be collected on the basis of timing – female flies
does not mate with the first 8 hour of emergence as adults.
A more efficient method for maximizing the number of virgins present in
the morning is to place the culture at 18 ℃ overnight, after your
collection at the end of the day. Development is slowed down
sufficiently at this temperature that there is roughly a 98% probability
that newly emerged females will mate for 16 hours. Thus, your morning
collections can be assumed to be virgins. Alternate 18 ℃ and 25 ℃.
Although male:female is 1:1, female flies tend to emerge earlier.
It is often convenient to store the flies you have collected and put 20 to
30 flies per vial. (Virgins should be used within 3 to 10 days). The best
timing is to make crosses at Fri. The first progeny will emerge 10 days
later, on Mon. During the storage period, it is also possible to ensure
that you have actual virgin by seeing that no larvae appear.
The P-element transposon
The P-element is much more commonly used in the fly community.
(a)
(b)
(c)
(d)
Tagging uncharacterized genes
Tagging a gene whose map position is well defined
Creating a new mutations with a P-element insertion
Creating deletions of a defined regions
A 2907-bp DNA fragment
31 bp repeat
Exon 1
Exon2
66 kD
▲
▲
AUG
Exon 0
poly A
termination
Exon 3
31 bp repeat
Transposase (87 kD from 2.7 kb mRNA)
The P-element Mediated Germ-line
Transformation
1. Preparation of DNA to be injected
Two UASG vectors:
Both vectors do not contain the start codon for
translation in region between the transcription
initiation and multiple cloning site. Therefore, the
start codon has to be put in when you are making
your P-element construct.
Kozak sequence: AA/CA/CATGGC
pUAST (Brand et al., 1993 development 118: 401)
pUC
3’ P
5’ P
white
BamHI  HindIII 5XUASG  hsp70TATA  EcoRI BglII NotI XhoIKpnI XbaI SV40 polyA BamHI
pUASpD (derived from pUASp MOD 78:113)
5’ P
3’ P
white
pUC
GAGA  7XUASG  XhoI  PTATA  KpnI NotI  BamHI BglII D-epitope  XbaI  K10 polyA  HindIII
KpnI
NotI
GG TAC Ccg ccc ggg atc aga tcc GCG GCC GCa tag gcc act agt gga tct
BamHI
BglII
XbaI
GGA TCC AGA TCT AGC CGT TCC GAG TCG AAG AGG AAT CGC TTC TAG A
D-epitope
Plasmid DNA to be injected has to be very clean.
CsCl- double banded
Column purified (endo-toxin free)- overload the column
PEG8000 precipitated
To 50 mg plasmid DNA - measured by Spectrophotometer
• add 5 mg of helper DNA
• precipitate by ethanol twice - 0.3 M NaOAc
• wash with 70% ethanol twice
• resuspend in an injection buffer (100 ml)
(0.1 M KPO4 pH 6.8, 5 mM KCl)
2. Collection of embryos, dechorionation and the injection
Set up a cage containing about 1000 flies
Mesh
w1118 ; 2-3 virgin females
w1118 males
For 2.5 liter
9-cm petri dish
Grape-juice plate
with a glob of yeast
paste at the center
67.5 g
500 ml
75 g
40 ml
agar
grape juice
sugar
10% Tegosept in ethanol
The first egg collection is discard because the first introduction of
fresh medium stimulates females to lay eggs that have been held
for a while.
The second egg collection can be used to test the injection
conditions, such as dryness of eggs and sharpness of needle.
There are three ways to remove chorion:
• cutting egg shell using tungsten or sharp dissecting needle
• gently rolling on double stick tape
• soaking in bleach (50-100%) for 3-5 minutes
After the chorion comes off, the anterior end can still be
identified easily by the presence of tiny micropyle at that end.
Dechorionated embryos are transferred onto a slide which has a
very thin strip of double stick tape on it. Embryos can be picked
up using a blunt dissecting needle or fine plastic fiber with a tiny
glob of gum at the tip. (A gum solution, sticky dip, can be made
by dissolving gum from double stick tape into heptane). The
needle or fiber can be dipped into the dip to coat a small amount
of gum on its tip. The embryos are orientated with their posterior
ends protruding over the edge of the tape.
Posterior ends of ebmryos
Double stick tape
~ 60 embryos
How many embryos lined up for one injection depends on humidity
of the room and personal skill. Embryos may need to be dessicated
in order to make them to take up the injected DNA. The best
condition in my lab is lining up ~60 embryos within 10 minutes and
wait for 2 more minutes when humidity is at 75%. Harlocarbon oil,
series 700, is then overlaid the embryos as thin as possible. They
are ready to be injected.
Injections are done with an inverted microscope and a
micromanipulator. We have used an air-filled system, in which
rubber tubing is hooked up a 50 cc plastic syringe attached to the
needle holder in the micromanipulator. The injection needle is
pulled from a 50 ml micropipet (Microcaps, Drummond; cat. # 1000-5000). The pull condition depends on individual needle puller.
The final diameter ranges from 2-6 mm. If the needle is too long, it
would not be strong enough to insert embryos.
If the needle is short, becoming wide too soon, the inserted part is
too fat to keep cytoplasm from leakage after the needle is
withdrawn. So, the final diameter, the sharpness of tip and the
taper of the needle are all important factors. Embryo stages!!!
After all embryos are injected, the slide is placed in a petri dish in
which a piece of round Whatmann filter paper has been taped on
the top and moistened with distilled water to keep the embryos in
humid when they develop. The incubation temperature is 18 ℃.
The survival larvae are transferred into vials containing the fly food.
Usually, each vial should have at least 50 larvae, which prevents
the food gone bad, but not more than 120. These larvae, kept at
25 ℃, would take 9 to 10 days to enclose. Adults are
backcrossed to w1118.
1
X
3-5 w1118
2
X
2 w1118
per vial
Pole cells
2
3
4
5
3. Screening of transformants and setting up lines
Eye color of transformants ranges from pale yellow to red.
This is a tendency that transformants with red eye color carry
multiple insertions. If one vial gives more than four
transformants, we just select four for the following procedures.
Amplification is carried out by backcrossing to w1118.
1 X 3-5 w1118
per vial
1118
1 X 2w
Progenies are used to set up two crosses:
1. Sibling cross
for determining the insertion on X and setup a line
2. Cross with a double balancer, y w; CyO/Sp; TM6B/Dr
for determining the insertion on 2nd and 3rd
Both w+ CyO and w+ Hu progenies are crossed with w1118
The GAL4/UAS system was used to
mis-express human genes in
Drosophila
Advantages:
• avoid lethality
•
can be induced
expression in various
tissues with one single
construct
•
various expression level
 different degrees of
severities
Brand et al., 1993 development 118: 401
An example: a mammalian snk
Overexpression of snk-GFPs in the eye imaginal disc
Phase
Three form of snks:
KM: kinase dead
wt: wild type
TE: kinase
consecutively active
wt
GFP
TE
KM
Ectopic snk expression results in rough eye
Controls: OR & GMR-GAL4
OR
GMR
KM
wt
TE
Ectopic snk expression results in loss of
rhabdomeres
GMR
TE
wt
KM
The kinase activity
is required
Isolate and identify modifers
1. Isolate mutants suppress or enhance the phenotype
2. Identify genes corresponding to the mutants
3. Characterize function of the genes
wt
eyg
wt
eyg