Drosophila Genetic Laboratory

PI: 廖國楨 /孫以瀚

Informationa. Books

Drosophila: A laboratory handbook The genome of Drosophila melanogaster The development of Drosophila melanogaster The embryonic development of Drosophila mel

anogaster Drosophila: A practical approach Fly pushing

b. The Flybase http://flybase.bio.indiana.edu mirror site http://flybase.nhri.org.tw

The Basics of Fly Genetics:

Fly ChromosomesFemale

MaleX 2L 3R3L2R 4

Drosophila stocks

1. Wild-type stocks: Oregon-R and Canton-S2. Mutant Stocks: Bloomington and Umea Stock Centers

Genetic map: e.g. 2-57 cM (centi-Morgan) No recombination in males

Y

Type of Mutations• Point mutation

• Inversion

• Translocation

• Compound chromosome

• Duplication

• Deficiency

( )

( )

Polytene chromosome

Physical mapping for chromosomal rearrangement, duplication and deficiency. X: 1-202L: 21-40 2R: 41-603L: 61-80 3R: 81-1004: 101-104

Fly Morphology

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)

Notum

Examples:

Eye shape and color

wild type Bar

BristleSco

Body colorBristle shape

Wing shape

wild type CyO

Sb and Hu

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 chr

omosome is homozygous• anything that is not shown is presumed to be wild type; simil

arly, 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.

Balancer Chromosomes

Features:• Homozygous lethal except FM• Multiple inversion• At least one dominant marker

inhibit recombinationfunction to trace chromosome and to maintain stock

FM: first multipleSM: second multipleTM: third multiple

Mating Scheme

FM7a CyOFM7a Sco X

ndY

or nd; multiple

FM7a CyO;nd +

virginfemales

Culture Condition

25 ℃ and 60% humidity

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% relativ℃e 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-agar2. Yeast-glucose-agar3. Instant potato medium4. Casein-based defined medium5. Yeast-cornmeal-molasses-agar6. Apple juice medium

Pests

1. Fungal control – Tegosept2. Bacterial control – penicillin G (less problem)3. Mite control -

(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

For new stocks:

Good housekeeping will help to reduce happening of mite infection:1. Autoclave all infected bottles and vials2. Clean your bench, microscope and fly handle equipments regularly3. Remove all old vials from fly room4. Use cotton plug instead of foam plug5. 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

Two etherizers and one apparatus to prevent food from falling

(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 stopp

ed moving.(e) Tip them on to the glass plate

for examination.

2. Carbon dioxide

An apparatus using CO2 fir anaesthetizing flies

Replace by a 16 G needle

(a) Tip flies into the anaesthetizer.

(b) Transfer to the porous ethylene film with CO2 the flowing through.

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 collectio℃n 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 i℃ ℃s 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) Tagging uncharacterized genes(b) Tagging a gene whose map position is well defined(c) Creating a new mutations with a P-element insertion(d) Creating deletions of a defined regions

A 2907-bp DNA fragment

▲ Exon 0 Exon2Exon 1 Exon 3

▲

Transposase (87 kD from 2.7 kb mRNA)

31 bp repeat

terminationpoly A

AUG

66 kD31 bp repeat

The P-element Mediated Germ-line Transformation

1. Preparation of DNA to be injected

pUAST (Brand et al., 1993 development 118: 401)

Two UASG vectors:

Kozak sequence: AA/CA/CATGGC

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.

white3’ P 5’ PpUC

BamHI HindIII 5XUASG hsp70TATA EcoRI BglII NotI XhoIKpnI XbaI SV40 polyA BamHI

GG TAC Ccg ccc ggg atc aga tcc GCG GCC GCa tag gcc act agt gga tct

GGA TCC AGA TCT AGC CGT TCC GAG TCG AAG AGG AAT CGC TTC TAG A

D-epitope

white3’ P pUC

GAGA 7XUASG XhoI PTATA KpnI NotI BamHI BglII D-epitope XbaI K10 polyA HindIII

pUASpD (derived from pUASp MOD 78:113)

5’ P

KpnI NotI

BamHI BglII XbaI

Plasmid DNA to be injected has to be very clean.

CsCl- double bandedColumn purified (endo-toxin free)- overload the columnPEG8000 precipitated

To 50 g plasmid DNA - measured by Spectrophotometer• add 5 g 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 injectionSet up a cage containing about 1000 flies

Mesh

Grape-juice platewith a glob of yeast paste at the center

For 2.5 liter 67.5 g agar500 ml grape juice75 g sugar40 ml 10% Tegosept in ethanol

w1118 ; 2-3 virgin femalesw1118 males or simply use w1118

9-cm petri dish

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.

Double stick tape

Posterior ends of ebmryos

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.

~ 60 embryos

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. # 1-000-5000). 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 . T℃he 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 backcrosse℃d to w1118.

1 X 3-5 w1118

2 X 2 w1118per 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

1 X 2 w1118per vial

Progenies are used to set up two crosses:

1. Sibling cross for determining the insertion on X and setup a line2. 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 Drosoph

ila

• avoid lethality

• can be induced expression in various tissues with one single construct

• various expression level different degrees of severities

Advantages:

Brand et al., 1993 development 118: 401

Phase

GFP

Overexpression of snk-GFPs in the eye imaginal disc

wt TE KM

Three form of snks:KM: kinase deadwt: wild typeTE: kinase consecutively active

An example: a mammalian snk

Ectopic snk expression results in rough eye

OR GMR

KM wt TE

Controls: OR & GMR-GAL4

GMR wt

TE KM

Ectopic snk expression results in loss of rhabdomeres

The kinase activity is required

Isolate and identify modifers

1. Isolate mutants suppress or enhance the phenotype2. Identify genes corresponding to the mutants3. Characterize function of the genes

wt eyg wt eyg