Hessian Fly Polytene FISH

Cytogenetic analysis using FISH in combination with polytene chromosome preparations is a powerful tool. Characterization of genetically modified insects can benefit greatly from this type of analysis.

Here is a protocol used for Hessian Fly Polytene FISH analysis provided by Jeff Stuart, Purdue University stuartjj@purdue.edu

Jeff Stuart

Jeff Stuart

HESSIAN FLY POLYTENE CHROMOSOME FISH

Slide Subbing

1) Subbing solution: 0.1% gelatin
0.01% chrom alum (CrK(SO4)2-12H2O) used as preservative).
To prepare, dissolve the ingredients, gelatin first, in ddH2O at 60˚C. Cool to room temperature before use. Store at 4˚C.
2) Immerse ethanol cleaned slides in subbing solution for a minute, remove slides, and allow to dry several hours before use. Alternatively, slides can be dried in 65˚C oven.

Chromosome Preparation

1) Dissect salivary glands from early to mid-staged 2nd instar larvae in 45% acetic acid. Several glands from several larvae should be collected for each slide.
2) Wash the salivary gland material by extracting the dissecting solution and replacing it with clean 45% acetic acid.
3) Place a single drop (~12 µl) of lactic-acetic acid (1 part lactic acid, 2 parts ddH2O and 3 parts glacial acetic acid) in the middle of a subbed slide. Transfer the salivary gland cells to this drop using a capillary tube. Transfer as little solution with the glands as possible.
4) Allow the glands to sit in the lactic-acetic acid for about 5 min. Then lower a siliconized, 18X18 mm coverslip (siliconized in Sigmacoat) over the drop on the slide taking care to prevent air bubbles from forming under the coverslip.
5) Carefully tap the edges of the slide against a piece of paper towel on the bench top. This knocks the chromosomes free of the nuclear membrane and the cytoplasm..
6) Check the condition of the chromosomes periodically under phase contrast (10 and 40X) but continue tapping until suitable spreading of the chromosomes is obtained.
7) Place the slide on a piece of bibulous paper or paper towel. Place another piece of absorbent paper over the slide. Using the ball of your hand, bare down on the coverslip with your weight to flatten the chromosomes. Take care not to move the coverslip- this will roll the chromosomes.
8) To flatten the chromosomes further, place the slide, coverslip down, between two paper towels sandwiched between two glass plates and leave a weight (brick) on top of the upper glass slide for at least 6 hours. If the slides are left for too long, the preparation may dry up. However, slides can be left to flatten overnight if the “sandwich” is performed at 4˚C.
9) Freeze the coverslip to the slide in liquid nitrogen or on a block of dry ice. Immediately flick off the coverslip with a razor blade and plunge the slide into 95% ethanol. Leave in 95% ethanol for at least 1 hour; it will not hurt to leave the slide overnight.
10) Air-dry in a dust-free atmosphere. The preparation is now stable and can be kept, preferable at 4˚C, in a dry, dust-free atmosphere before hybridization.

Preparing Slides for Hybridization

1) Heat slides for 30 min. in 2X SSC at 65˚C.
2) Wash slides for 2 min. in 2X SSC at room temp.
3) Dehydrate in EtOH series of 5 min. washes: 70%, 90%, 95%, and 100% EtOH.
4) Allow the slides to air-dry.
5) Denature chromosomes by incubating in freshly prepared 0.07 N NaOH for 3 min. (0.7 g in 250 ml ddH2O, or 0.35 ml 10 N NaOH in 50 ml ddH2O).
6) Wash slides in 2X SSC for 5 min.
7) Dehydrate in EtOH series of 5 min. washes: 70%, 90%, 95%, and 100% EtOH.
8) Air dry thoroughly. At this point, the slides are ready to hybridize.

PROBE PREPARATION

Nick translation of 1µg of BAC clone probe DNA.

1) Preparation of BAC clone DNA- PSI Clone BAC DNA Kit for 3-5 ml cultures (Princeton Separations, Inc. 800-223-0902, http:www.prinsep.com, cat. no. PP-120)

2) Nick translation reactions for biotin probes:

Suggested chemicals: Nick Translation Kit, Enzo Diagnostics, Cat. No. 42803 (800-221-7705)

10X polymerase buffer 5 µl
0.5 mM dNTP 5 µl
biotin-dUTP mix 5 µl
1:5 DNase I 1 µl
DNA polymerase I 1 µl
DNA template (~1 µg) 10 µl
ddH2O) 23 µl
Total 50 µl

3) Nick translation reactions for digoxygenin probes:

Digoxygenin Nick Translation mix (Roche) 4 µl
DNA template (~1 µg) and ddH2O 16 µl
Total 20 µl

4) Incubate reactions at 15˚C for 2 h.

5) Stop reactions by adding 1/10th volume of 0.2 M EDTA.

6) Clear reactions with Qiagen QIA Quick Nucleotide Removal Kit and elute DNA with 50 µl of elution buffer.

7) Use 1 to 2 µl of probe (20-40 ng) in the hybridization mixture described below.

HYBRIDIZATION

1) Probe mixture per slide: Dry each probe DNA (20 to 40 ng each) and dissolve in 1µl of ddH2O.

Hybridization Mixture

Probe DNA (20-40 ng) x µl
10 mg/ml salmon sperm DNA 1 µl
20X SSC 1 µl
Deionized formamide 5 µl
50% dextran sulfate 2 µl
Total 10 µl

2) Denature probe mixture at 80-90˚C for 5 to 15 min.

3) Place mixture on ice.

4) Place 10 µl of probe mixture on slide and cover with 22x22mm coverslip. Seal with rubber cement.

5) Place slide in a humid box at 37˚C overnight.

Detection

1) Gently remove rubber cement

2) Place sides into room temperature 2X SSC to allow coverslip to fall away.

3) Wash in 2X SSC at room temp. for 5 min.

4) Wash in 2X SSC at 42˚C for 10 min.

5) Wash in 2X SSC at room temp. for 5 min.

6) Wash in 1X PBS at room temp. for 5 min.

7) Incubate with 100 µl of antibody mix under a 22×44 mm coverslip for 30 min. at 37˚C.

Antibody mix per slide

5X Antibody buffer (5X PBS, 5% BSA) 20 µl
Anti-digoxigenin 1 µl
Anti-biotin 1 µl
DdH2O 78 µl
Total 100 µl

8) Wash 3-times with 1X PBS at room temp. for 5 min. each time.

9) Add 12 µl of Vectasheild with DAPI counterstain and cover with a 22×44 coverslip. Squash preparation gently.

BIOTIN LABELED IN SITUS (cont.)

Solutions

PBS: 1X PBS 10X Mix
NaCl 130 mM 75.97 g
Na2HPO4-2H2O 7mM 12.46 g
NaH2PO4-2H2O 3 mM 4.8 g

To prepare 10X solution, mix salts together in less than 1L, adjust to pH 7.0, raise volume to 1L with ddH20. Store at room temp.

TMN1, 0.05% Triton X-100 Final Mix (1L)
Tris-HCl (pH 7.5) 100 mM 100 ml of 1 M
NaCl 100 mM 20 ml of 5 M
MgCl2 2 mM 1 ml of 2 M
distilled water to less than 1 L
Sterilize by autoclaving; when cooled add 0.5 ml Triton X-100 and raise volume to 1L with ddH20. Store at 4˚C.

2% BSA in TMN1, Triton X-100: Dissolve 2 g BSA/100 ml of TMN1, Triton X-100 buffer)

TMN2: Final Mix (1L) Mix (200 ml)
Tris-HCl (pH 9.0) 100 mM 100 ml of 1 M 20 ml of 1 M
NaCl 100 mM 20 ml of 5 M 4 ml of 5 M
MgCl2 50 mM 25 ml of 2 M 5 ml of 2 M
Add distilled water to appropriate volume and autoclave. Store at room temp.

Recommended that all buffers be filtered before use to reduce background problems.

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