Karyotyping Biotechnology I Powerpoint modified from powerpoint provided by NIH (Mark Nardone and Roland Nardone, authors)

Essential Question How do we use human cell cultures to look for abnormalities in chromosome structure?

Human Cells Have 23 pairs of chromosomes or 46 total One of the pair donated by the biological mother The other donated by the biological father

Karyotyping-Definition Physical Characterization of chromosomes Examine diploid chromosomes for Size Shape Look for structural change which reveals abnormality As part of physical characterization take a photomicrograph of the chromosomes either digitally or physically

Karyotyping The photomicrograph is cut Chromosome pairs are lined up 1-23 Look for abnormalities

Purpose of Karyotyping Serves many areas of basic biology and medicine including Species identification Gene mapping- where genes are located on the chromosome Genetic disease analysis

Species Identification Each species has a characteristic karyotype Humans Mouse

Gene Mapping Genes are mapped to specific chromosomes Each chromosome has a unique banding pattern based of staining more or less condensed portion of DNA Banding patterns are species specific

Inherited Genetic Diseases Many arise from changes in chromosomes Changes can be Number of chromosomes- Ex Down’s Syndrome – 3 of chromosome 21

Inherited Genetic Diseases Structural changes Ex. Cri du Chat Syndrome –deletion of short arm of chromosome 5

Human Karyotyping Test used to identify chromosome aberrations Used as part of pre-natal diagnosis Cells collected Amniocentesis Physician insert needle into uterus and collect 10 mL of amniotic fluid Occurs 14th-16th week of pregnancy Chorionic villus Physician inserts narrow tube into cervix and sucks tiny sample of tissue from placenta Can be performed 8th-10th week of pregnancy

Sources of Cultured Cells for Karyotyping. S Source Purpose Lymphocytes from peripheral blood Clinical Diagnosis Cultured fibroblasts collected by amniocentesis or chorionic villus sampling Prenatal Diagnosis Established cell lines from human or rodent: IMR 90 Human Lung Fibroblast Chinese Hamster Ovary Cells T3 Mouse Prostate Cells Research

Cells in Lab today Human tumor cell line –HeLa cells Originated from cervical cancer cells of Henrietta Lacks Because cells are of tumor origin, divide continuously Do not contain the normal diploid number of chromosomes Greater than 23 pairs

Cell Cycle in Eukaryotes Alternates between two phases Interphase=non-dividing phase Cell grow Cell metabolically active-synthesize macromolecule During this phase chromosomes duplicated in preparation for division Represents 90% of life cycle M-phase=dividing phase Shortest part of life cycle. Two events occur Mitosis – nuclear division Duplicated chromosomes separate Cytokinesis-cytoplasm division

Cell Cycle in Eukaryotes End result two cells with same number of chromosomes that are genetically identical

M Phase Mitosis-nuclear division Duplicated chromosomes separate Divided into five stages Prophase, prometaphase, metaphase, anaphase, telophase (PPMAT) For karyotyping freeze the cells in metaphase where chromosomes highly condensed and visible

Metaphase Blockade Purpose is to increase the number of cells in metaphase Accomplished by adding the drug, colchicine Colchicine block cells from going from metaphase to anaphase

Hypotonic Treatment of Metaphase Blocked Cells Hypotonic salt solution (0.075M KCl). Causes cell swelling, cells are more fragile, rupture easily when splattered onto slides.

Application of Fixed Cells to Slide Dispense cell suspension, dropwise, from a fine bore pipette onto a clean slide. The pipette tip should be at least 12 inches (3 feet is best!) above the slide. The slide should be propped up at a 45O angle. Allow slide to air dry while blowing over it. Spreading of chromosomes is enhanced by rapid evaporation of the fixative.

Staining the Chromosome Spreads After cells are fixed on the slide, they will be stained with stain 1 followed by stain 2 A 2 drops of paramount will be applied to stained area A glass coverslip will be placed on top of paramount Gently apply pressure to the coverslip to spread the paramount evenly under coverslip May want to place two coverslips side by side to view entire microscope slide

Search for Ideal Chromosome Spread Key Characteristics Search for Ideal Chromosome Spread Key Characteristics No overlapping chromosomes Chromosomes easily counted Arms of chromosomes readily visualized Make photographs of chromosome spread

Human Chromosome Spread