Lesson 9 Flashcards
Cytogenic techniques, Genetic Engineering, & Gene Therapy
The study of chromosomes using traditional cytogenetic techniques requires cells that are actively dividing
Cytogenetic Technique
Chromosomes are individually distinguishable under _____ microscope only during cell division and are best examined during ________
Light microscope; metaphase
Metaphase chromosomes can be obtained from specimens that contain
spontaneously dividing cells
cultured
and chemically induced to divide in vitro
Specimens that contain spontaneously proliferating cells include:
- bone marrow
- lymph nodes
- solid tumors
- chorionic villi
What are routinely cultured to obtain dividing cells
Peripheral blood lymphocytes
tissue biopsies
amniotic fluid samples
Lymphocytes usually require
The additional of a mitotic stimulant
It depends on clinical indications and whether the diagnosis is prenatal or postnatal
The choice of specimen for chromosome analysis
The most critical requirement of culture initiation, maintenance, and cell harvest
Living cells capable of cell division must be received by the laboratory
The laboratory may reject specimens that are
improperly labeled or unlabeled
Living cells
Sterility
Proper growth medium
+/- Mitotic stimulant
Microbial inhibitors
Culture Initiation
Sterility
Optimal temperature
Optimal pH
Optimal humidity
Optimal time interval
Culture Maintenance
Arrest division
Swell cells
Fix cells
Prepare slide
Stain/band
Cell Harvest
2 broad categories of Chromosome banding and staining
- Produce specific alternating bands along the length of each entire chromosome
- Stain only a specific region of some or all chromosomes
This creates unique patterns for positive identification and permits characterization of structural abnormalities; numerical and structural examination
Specific alternating bands
Used in special circumstances when a particular piece of information cannot be answered using a routine banding method
Stain only a specific region
Techniques that create bands along the length of the chromosomes
G-banding
Q-banding
R-banding
Techniques that stain selective chromosome regions
C-banding
T-banding
Cd-banding
G-11 banding
NOR
DAPI/DA Staining
FISH
G bands produced with trypsin and Giemsa
GTG banding
dark bands in G-banding
A-T rich (late replicating, heterochromatic regions)
light bands in G-banding
C-G rich (early replicating, euchromatic regions)
The biologically more significant ACTIVE REGIONS
G-light bands
The relatively few active genes
G-dark bands
other stains used in G-banding techniques
(Giemsa)
Wright’s
Leshman’s
Fluorescent technique that was first developed for human chromosomes
Q-banding
What certain fluorochromes will bind to DNA and produce distinct banding patterns of bright and dull fluorescence when excited with the proper wavelength of light
quinacrine dihydrochloride
The brightly fluorescing regions are what in Q-banding
A-T rich
What differentiates Q-banding from G-banding?
distal long arm of the Y fluoresce brightly
Produce a banding pattern that is the OPPOSITE OR REVERSE of the G-banding pattern
R-banding
Methods in R-banding
fluorescent
nonfluorescent
R-banding C-G rich
heterochromatic regions that stain darkly (bright)
R-banding A-T rich
euchromatic regions that stain lightly (dull)
What is the positive region in R-banding?
light stained, fluoresce dull, A-T rich euchromatic
C-banding means
Constitutive Heterochromatin Banding
Where does C-banding selectively stain?
the constitutive heterochromatin around the centromeres
Useful for determining the presence of dicentric and pseudodicentric chromosomes = marker chromosomes and polymorphic variants
C-banding
Resists degradation
Constitutive heterochromatin
Selectively depurinated and denatured by barium hydroxide
CBG banding
Results in only the terminal ends or TELOMERES
T-banding
Pair of dots at each centromere, one on each chromatid that differentiate functional from nonfunctional centromeres
Cd staining
Stains the pericentromeric regions of all chromosomes at high alkaline pH to delineate heterochromatin polymorphisms
G-11 banding
Selectively stains silver nitrate on the nucleolar organizer regions located on the satellites stalks of the acrocentric chromosomes
NOR
Theoretically, how many NORs are per cell and, for each acrocentric chromosome?
10 NORs, 1 for each acrocentric chromosome
Combines DAPI (fluorescent dye) w/ distamycin A (nonfluoro antibiotic) to certain A-T rich areas of constitutive heterochromatin in C-band regions for differentiating between satellite regions of any of the acrocentric chromosomes
(1,9,16 distal, short arm 15)
DAPI/DA staining
(4,6-diamino-2phenole-indole/ distamycin A)
Uses fluorescent probes that attaches to specific areas in the chromosome with high degree of sequence complementarity
FISH
Used to detect and localize presence or absence of specific DNA sequences in the chromosomes to determine the possible cause of a child’s developmental disability
FISH
Collection of microscopic DNA spots (traditional solid/alt bead array)
DNA microarray analysis
Manipulation of an organism’s nucleic acids or the insertion of DNA into cells
Genetic engineering
One of whose genes have been artificially manipulated for a desired effect
Genetically Modified Organisms (GMO)
Technology used to cut a known DNA sequence from one organism and introduce it into another organism thereby altering the genotype and therefore altering the phenotype of the recipient
Recombinant DNA technology
Process of isolating genes from one organism, manipulating purified DNA in vitro, and transferring to another organism
Gene cloning
Who started gene cloning when discovering enzymes degrading bacterial viruses
Arber (1950)
Who purified enzymes and characterized them to be able to cut DNA at specific sites?
Smith (1970)
Purified enzymes that recognizes a specific sequence of bases and cut the DNA backbone
Restriction enzymes (restriction endonucleases)
Recombinant vectors
-plasmids
-specially designed bacteriophage lambda chromosomes
-cosmids
the cut DNA is introduced into
the plasmid
Restriction enzymes acts as endonucleases to cleave a DNA sequence at a specific site called
Restriction site (recognition sequence)
Plasmid containing the cloned gene
Chimera
The DNA sequence that has been incorporated in the vector
Insert
Methods of inserting DNA into a cell
- heat shock method
- electroporation
- genetically engineered viruses
- gene gun
- microinjection
- liposomes
When chimera plasmids are placed in a solution (w/ cold CaCl2 + normal host bacteria) with ABRUPT HEATING at 42°C for 2-5 minutes
Heat shock method
When host cells are exposed to HIGH VOLTAGE PULSE
Electroporation
Genetic engineering applications
- therapeutic applications
- gene therapy
- chimeric monoclonal antibodies
- HGP/ Human Proteome Project
- Scientific applications
Experimental technique that utilizes genes to treat or prevent diseases by inserting a gene into a patient’s cells instead of using medicines/drugs or surgery
Gene therapy
Two main types of gene therapy
- Somatic cell
- Reproductive or germ-line
Genes are introduced in an organ or tissue to induce production of specific products
DOES NOT AFFECT the individual’s GENETIC MAKEUP as a whole (transmitted to the next)
Allows damaged organs to function properly
Somatic cell gene therapy
Integration of corrective genes into sex cells or zygotes with the goal of generating beneficial genetic change that CAN BE TRANSMITTED to the progeny
Offspring can inherit the introduced genes
Reproductive or germ-line gene therapy
Medical conditions for which gene therapy is being investigated on:
- bubble boy disease
- hemophilia
- AIDS
- cancer (liver, lung. breast, colon, ovarian, prostate)
- asthma
- brain tumor
- melanoma
- muscular dystrophy
- neurodegenerative conditions
- diabetes
- heart diseases
