KARYOTYPING.


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CYTOGENETICS. Is the investigation of the structure and properties of chromosomes, chromosomal conduct amid mitosis and meiosis, chromosomal impact on the phenotype and the variables that cause chromosomal changes (Hare and Singh, 1979). . Classification of chromosomes. . . . Readiness OF CHROMOSOMES. .
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KARYOTYPING Dr. R.A. Siddique M.V.Sc PhD Scholar National Dairy Research Institute Karnal, (Haryana) 132001 India

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CYTOGENETICS Is the investigation of the structure and properties of chromosomes, chromosomal conduct amid mitosis and meiosis, chromosomal impact on the phenotype and the variables that cause chromosomal changes (Hare and Singh, 1979).

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Nomenclature of chromosomes

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PREPARATION OF CHROMOSOMES

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METHODOLOGY Aseptic safety measures Preparation of RPMI 1640 medium Collection of 10ml of blood with heparin Setting of culture 8 ml of medium 0.1 ml of PHA-M 0.5 ml of blood/plasma 2 ml of autologus plasma/FCS Incubate at 37  C for 72 hours

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METHODOLOGY… Harvesting of culture Spindle inhibitors – Colchicine/colcemed (0.1 g/ml) Hypotonic treatment – 0.075M KCl Fixation (3:1 methanol : acidic corrosive) Preparation of Slides recolored with 4% Giemsa for 20-25min Screening of slides to concentrate on the morphology of chromosome Construction of karyotype

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MITOTIC CHROMOSOMAL SPREAD OF CATTLE

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MITOTIC CHROMOSOMAL SPREAD OF CATTLE

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NORMAL KARYOTYPE OF CATTLE

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TERMS AND DEFINITIONS OF VARIOUS ABERRATIONS OF CHROMOSOMES Ring( r) Minute (min) Dicentric (d) Hyperdiploid (h) Chromosome crevice (sg) Chromatid cancellation (td) Fragment (f) Acentric piece (af) Translocation (t) Triradial (tr) Quadriradial (qr) Pulverized chromosome (pu) Pulverized chromosome (pu+) Pulverized cell (puc) Complex revision (cr) Polyploid (pp) or endoreduplication

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BANDING OF CHROMOSOMES G - Banding Q - Banding C - Banding R - Banding T - Banding NOR - Banding High Resolution Banding Restriction Endonuclease Banding

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Q-banding  1. Get dried out the slides by plunging in liquor with diminishing focus 90%, 70% and half one min each. 2. Flush in refined water. . 3. Wash the slide in phosphate cushion at pH 6.8. 4. Recolor the slide in quinacrine mustard (5 mg in 100 mI) or in quinacrine dihydrochloride 5% for 20 min. 5. Flush in phosphate cradle and mount in a similar cushion. 6. Inspect under fluorescent magnifying lens.

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C-banding 1. Treat the slides in 0.2 N HCI for one hr at room temperature. 2. Wash in de-ionized water. 3. Submerge in 1% barium hydroxide at 50°C for 5-15 min. 4. Flush in deionized water. 5. Brood at 60°C in 2XSSC cushion for one hr. 6. Wash in de-ionized water and stain in 4% Giemsa recolor for 90 min. 7. Wash in de-ionized water, dry and look at under oil inundation.

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R-banding 1. Age the slides for 7 - 10 days . 2. Put the slides in a Coplinjar containing phosphate cushion ofpH 6.5 at 85°C and brood for 20-25 min. 3. Recolor the slides in 0.01% acridine orange in the phosphate support pH 6.5 for 4-6 min. Flush in phosphate support and mount in a similar cushion. 4. Analyze under fluorescent magnifying lens.

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T - banding 1. Age the slide for 7 days. 2. Place.the slides in PBS pH 5.0 for 20-60 min at 87°C. 3. Flush in PBS. 4. Recolor in 3% Giemsa in phosphate cushion pH 6.8 at 87°C, leave for 5-30 min and flush. 5. Slides are recolored in Hoechst 33258 stain for 10 min (Hoechst recolor 0.5 pg/m1 of phosphate buffer).Rinse in phosphate support and look at in fluorescent magnifying lens. 6. On the other hand, the recolored slides are secured with a cover slip and set in a wet chamber under UV light for 2 to 3 hrs or under direct daylight for 2 hrs. 7. Expel the cover slip and stain in Giemsa recolor for 10 min. 8. Wash in support, dry and mount in DPX.

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METHODOLOGY G-Banding procedure Aging of good slides for 10 days Normal saline Treated with trypsin 0.25% arrangement 10-15 sec Immersed in 70% ethanol for couple of minutes Stained with 10% Giemsa for 6-10min Microphotograph great spreads Construction of G-grouped karyotype

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G-BANDED MITOTIC CHROMOSOMAL SPREAD OF CATTLE

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G-BANDED KARYOTYPE OF CATTLE

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THANX FOR UR KIND ATTENTION

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