Dr. R.A. Siddique

M.V.Sc PhD Scholar

National Dairy Research Institute

Karnal, (Haryana) 132001

India

CYTOGENETICSCYTOGENETICS

Is the study of the structure and properties of Is the study of the structure and properties of chromosomes, chromosomal behaviour during chromosomes, chromosomal behaviour during mitosis and meiosis, chromosomal influence on mitosis and meiosis, chromosomal influence on the phenotype and the factors that cause the phenotype and the factors that cause chromosomal changes (Hare and Singh, 1979). chromosomal changes (Hare and Singh, 1979).

Nomenclature of Nomenclature of chromosomeschromosomes

PREPARATION OF PREPARATION OF CHROMOSOMESCHROMOSOMES

METHODOLOGYMETHODOLOGY Aseptic precautionsAseptic precautions Preparation of RPMI 1640 mediumPreparation of RPMI 1640 medium Collection of 10ml of blood with heparinCollection of 10ml of blood with heparin Setting of cultureSetting of culture

8 ml of medium8 ml of medium0.1 ml of PHA-M0.1 ml of PHA-M0.5 ml of blood/plasma0.5 ml of blood/plasma2 ml of autologus plasma/FCS2 ml of autologus plasma/FCS

Incubate at 37Incubate at 37C for 72 hoursC for 72 hours

METHODOLOGY…METHODOLOGY…

Harvesting of cultureHarvesting of culture Spindle inhibitors – Colchicine/colcemed Spindle inhibitors – Colchicine/colcemed

(0.1(0.1g/ml)g/ml) Hypotonic treatment – 0.075M KClHypotonic treatment – 0.075M KCl Fixation (3:1 methanol : acetic acid)Fixation (3:1 methanol : acetic acid) Preparation of slidesPreparation of slides Slides stained with 4% Giemsa for 20-Slides stained with 4% Giemsa for 20-

25min25min Screening of slides to study the Screening of slides to study the

morphology of chromosomemorphology of chromosome Construction of karyotypeConstruction of karyotype

MITOTIC CHROMOSOMAL SPREAD MITOTIC CHROMOSOMAL SPREAD OF CATTLEOF CATTLE

MITOTIC CHROMOSOMAL SPREAD OF MITOTIC CHROMOSOMAL SPREAD OF CATTLECATTLE

NORMAL KARYOTYPE OF CATTLE

TERMS AND DEFINITIONS OF VARIOUS TERMS AND DEFINITIONS OF VARIOUS ABERRATIONS OF CHROMOSOMESABERRATIONS OF CHROMOSOMES

Ring( r)Ring( r) Minute (min)Minute (min) Dicentric (d) Dicentric (d) Hyperdiploid (h)Hyperdiploid (h) Chromosome gap (sg)Chromosome gap (sg) Chromatid deletion Chromatid deletion

(td)(td) Fragment (f)Fragment (f) Acentric fragment (af)Acentric fragment (af) Translocation (t)Translocation (t) Triradial (tr)Triradial (tr) Quadriradial (qr)Quadriradial (qr) Pulverized Pulverized

chromosome (pu)chromosome (pu) Pulverized chromosome (pu+)Pulverized chromosome (pu+) Pulverized cell (puc)Pulverized cell (puc) Complex rearrangement (cr)Complex rearrangement (cr) Polyploid (pp) or endoreduplicationPolyploid (pp) or endoreduplication

BANDING OF BANDING OF CHROMOSOMESCHROMOSOMES

G - BandingG - Banding Q - BandingQ - Banding C - BandingC - Banding R - BandingR - Banding T - BandingT - Banding NOR - BandingNOR - Banding High Resolution BandingHigh Resolution Banding Restriction Endonuclease Restriction Endonuclease

Banding Banding

Q-bandingQ-banding

  1. Dehydrate the slides by dipping in 1. Dehydrate the slides by dipping in alcohol with decreasing concentration alcohol with decreasing concentration 90%, 70% and 50% one min each.90%, 70% and 50% one min each.

2. Rinse in distilled water. .2. Rinse in distilled water. .3. Wash the slide in phosphate buffer at pH 3. Wash the slide in phosphate buffer at pH

6.8.6.8.4. Stain the slide in quinacrine mustard (5 4. Stain the slide in quinacrine mustard (5

mg in 100 mI) or in quinacrine mg in 100 mI) or in quinacrine dihydrochloride 5% for 20 min.dihydrochloride 5% for 20 min.

5. Rinse in phosphate buffer and mount in 5. Rinse in phosphate buffer and mount in the same buffer.the same buffer.

6. Examine under fluorescent microscope.6. Examine under fluorescent microscope.

C-bandingC-banding

1. Treat the slides in 0.2 N HCI for one hr 1. Treat the slides in 0.2 N HCI for one hr at room temperature.at room temperature.

2. Rinse in de-ionized water.2. Rinse in de-ionized water.3. Immerse in 1% barium hydroxide at 3. Immerse in 1% barium hydroxide at

50°C for 5-15 min.50°C for 5-15 min.4. Rinse in deionized water.4. Rinse in deionized water.5. Incubate at 60°C in 2XSSC buffer for 5. Incubate at 60°C in 2XSSC buffer for

one hr.one hr.6. Rinse in de-ionized water and stain in 6. Rinse in de-ionized water and stain in

4% Giemsa stain for 90 min.4% Giemsa stain for 90 min.7. Rinse in de-ionized water, dry and 7. Rinse in de-ionized water, dry and

examine under oil immersion.examine under oil immersion.

R-bandingR-banding1. Age the slides for 7 -10 days .1. Age the slides for 7 -10 days .

2. Place the slides in a Coplinjar 2. Place the slides in a Coplinjar containing phosphate buffer ofpH 6.5 at containing phosphate buffer ofpH 6.5 at 85°C and incubate for 20-25 min.85°C and incubate for 20-25 min.

3. Stain the slides in 0.01% acridine 3. Stain the slides in 0.01% acridine orange in the phosphate buffer pH 6.5 orange in the phosphate buffer pH 6.5 for 4-6 min. Rinse in phosphate buffer for 4-6 min. Rinse in phosphate buffer and mount in the same buffer.and mount in the same buffer.

4. Examine under fluorescent microscope.4. Examine under fluorescent microscope.

T -bandingT -banding

1. Age the slide for 7 days.1. Age the slide for 7 days.2. Place.the slides in PBS pH 5.0 for 20-60 min at 87°C.2. Place.the slides in PBS pH 5.0 for 20-60 min at 87°C.3. Rinse in PBS.3. Rinse in PBS.4. Stain in 3% Giemsa in phosphate buffer pH 6.8 at 87°C, 4. Stain in 3% Giemsa in phosphate buffer pH 6.8 at 87°C,

leave for 5-30 min and rinse.leave for 5-30 min and rinse.5. Slides are stained in Hoechst 33258 stain for 10 min 5. Slides are stained in Hoechst 33258 stain for 10 min

(Hoechst stain 0.5 pg/m1 of phosphate buffer).Rinse in (Hoechst stain 0.5 pg/m1 of phosphate buffer).Rinse in phosphate buffer and examine in fluorescent phosphate buffer and examine in fluorescent microscope.microscope.

6. Alternatively, the stained slides are covered with a cover 6. Alternatively, the stained slides are covered with a cover slip and placed in a wet chamber under UV lamp for 2 to slip and placed in a wet chamber under UV lamp for 2 to 3 hrs or under direct sunlight for 2 hrs.3 hrs or under direct sunlight for 2 hrs.

7. Remove the cover slip and stain in Giemsa stain for 10 7. Remove the cover slip and stain in Giemsa stain for 10 min.min.

8. Rinse in buffer, dry and mount in DPX.8. Rinse in buffer, dry and mount in DPX.

METHODOLOGYMETHODOLOGYG- Banding techniqueG- Banding technique Ageing of good slides for 10 daysAgeing of good slides for 10 days Normal salineNormal saline Treated with trypsin 0.25% solution 10-15 secTreated with trypsin 0.25% solution 10-15 sec Immersed in 70% ethanol for few minutesImmersed in 70% ethanol for few minutes Stained with 10% Giemsa for 6-10minStained with 10% Giemsa for 6-10min Microphotograph good spreadsMicrophotograph good spreads Construction of G-banded karyotypeConstruction of G-banded karyotype

G-BANDED MITOTIC CHROMOSOMAL G-BANDED MITOTIC CHROMOSOMAL SPREAD OF CATTLESPREAD OF CATTLE

G-BANDED KARYOTYPE OF CATTLE