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

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

3. 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

4. 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

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

6. 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

7. Species Identification
Each species has a characteristic karyotype
Humans
Mouse

8. 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

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

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

11. 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

12. 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

13. 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

14. 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

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

16. 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

17. 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

18. 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.

19. 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.

20. 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

21. 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

22. Human Chromosome Spread