Transcript LATg Training Course - AZ Branch AALAS Homepage

LATG
Chapters 8 & 9
Molecular Biology and Genetics
Molecular Biology
• …is the study of biology at the
molecular level
• …focuses specifically on DNA, RNA,
and protein
• …is a tool used to study genetics
Some Definitions
• Genetics …is the study of how
genes interact (with each other AND
their environment) to produce the
inherited characteristics that we see
every day
• Genome …the entire collection of
genes an organism has.
More Definitions:
• Genotype = the genetic makeup of an
organism
– Every person (every mouse, every cow) has two
copies of each gene, one from each parent
• “Homozygous normal” = two normal copies
(aka
Wildtype)
• “Heterozygote” = one normal & one abnormal copy
• “Homozygous abnormal” = two abnormal copies (in
transgenics, aka “Knock-Out”
There’s still more...
• Phenotype = the physical features of an
organism (i.e., tall/short; red/white etc)
• Mutation = any change in the DNA of a
gene
• Genetic Engineering...is the term used
to describe the manipulation of the
genetic make-up of an organism
Where is your DNA located?
Chromosomes from a human female
Where is your DNA?
• DNA is in the nucleus of the cell, on
structures called chromosomes.
• Chromosomes are made of genes
• Genes are made of DNA
Structure of DNA
• DNA is a long string (polymer) of 4 bases
• These bases universal!
– A = Adenosine
– T = Thymine
– C = Cytosine
– G = Guanine
• The order (sequence) of the bases is what
makes one gene different from another gene.
DNA Structure, cont’d
• A -- T
• C -- G
• GATTCC
CTAAGG
• DNA exists as a double helix
(twisted ladder)
• Each rung of the ladder is a
base pair
How do cells transmit their
genetic information?
• Replication = the
process by which a
cell duplicates its
DNA.
• When a cell
divides,one copy
gets passed onto
the new cell
How do cells interpret the
information in the DNA?
• Transcription: the process by which the
DNA code is “read”.
– DNA is transcribed into RNA in the nucleus
Transcription
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•
•
•
•
RNA has 4 bases:
Adenine
A=U
Guanine
G=C
Cytosine
Uracil*** (Uracil is used instead of
Thymine)
• Unlike DNA, RNA is single-stranded
Translation
• Occurs outside the
nucleus, via
ribosomes
• RNA is “read” in
groups of three
bases called
“codons.”
• Each codon
corresponds to an
amino acid
Information Flow in the Cell
Techniques
•
•
•
•
•
Extraction of DNA
Restriction Digestion
PCR
Electrophoresis
Southern blotting
Extraction of DNA
• Enzymes “digest”
cell walls and
release DNA into
solution
• Add phenol to
remove proteins
• Spin to separate
DNA from proteins
• Add ethanol
Restriction enzymes
• These are enzymes that cut (digest) DNA at
specific sites (sequences).
• Examples:
– Eco RI only cuts the sequence
…GAATTC…
– Pst 1 only cuts the sequence …CTGCAG
• Because everyone’s DNA is comprised of the
same 4 nucleotides (A,T, C, G), you can
attach one species to another...
Cloning (using RE)
Mouse
Bacteria
CGAAGGAATTCCGTAG
GCTTCCTTAAGGCATC
TGATTGAATTCTTAAC
ACTAACTTAAGAATTG
+ Eco R1
CGAAG
GCTTCTTAA
AATTCCGTAG
GGCATC
TGATTG
AATTCTTAAC
ACTAACTTAA
GAATTG
+ ligase (another enzyme that attaches the m/c)
CGAAG AATTCTTAAC
GCTTCTTAAGAATTG
Polymerase Chain Reaction
(PCR)
• Based on the fact that A=T and C=G
• Need only a tiny bit of DNA
• ***Must know some of the sequence of the
gene of interest
• Three simple steps: heat, add primers etc,
cool solution so bases will bind, repeat!
• Can amplify a piece of DNA a million-fold!
PCR
Detection of DNA
• Use agarose gels
• Gel acts like a filter: DNA separates by
size
• Stain the gel with a dye to make the
DNA to make it fluoresce under UV
light.
Blotting
• Southern Blot
– DNA cut with enzymes
– transferred to membrane
– hybridized to probe that is specific to gene
of interest
– exposed to film
– if gene is present, you will see a band on
the film
Types of Blots
• Southern blotting -- DNA
• Northern blotting -- RNA
• Western blotting -- protein
Genetic Engineering of Mice
– Two types
• Transgenic - a gene is added via pronuclear
injection
– This is used to “overexpress” a gene
– ex: Alzheimer’s and Beta-amyloid
• Targeted Mutation (aka “KO”) - a gene altered
then added to the genome using ES cells
– This is used to delete a gene
– ex: ERKO--estrogen receptor KO mice
Transgenic mice
• Created through pronuclear injection
• Need 4 groups of mice
– superovulated females
– stud males
– vasectomized males
– pseudopregnant females
The Mice
• Superovulated females --given hormone
injections to make them release more eggs
than usual (30-60)
• Stud Males --are mated with the s.o.females
so that a lot of embryos are produced
• Pseudopregnant females --a female is mated
to a sterile male so that her body will produce
hormones that prepare it for pregnancy
Procedure
Transgenic Mice
• Transgene can integrate ANYWHERE
in the mouse genome.
• Integrates in 1-several hundred copies
• Must screen pups (PCR) to determine
which pup have the transgene, and will
pass it on.
• Must observe transgenic mice carefully
to observe phenotype
Phenotype
• Depends on the
gene you’re
overexpressing
• Sometimes it’s
obvious, sometimes
it’s not.
• Must observe these
mice carefully
Knockout Mice
• Knockout mice --a gene is deleted
• Similar to transgenic mice, must
carefully observe for phenotype
• Need the same 4 groups of mice, plus
ES cells
• ES cells = embryonic stem cells
– totipotent
Embryonic Stem Cells
• Are from the inner
cell mass of a
blastocyst
• can develop into any
part of the body
• “totipotent”
KnockOut Mice
• Culture ES cells from white (129) mice
and target gene using electric current
• Mate black mice; insert “white” ES cells
into black blastocyst
• Pups = chimeras (black and white)
• Mate chimera to black mouse.
• If white pups are produced, targeted
gene has been passed on!
Transgenic Animals and You
• You are very important!!
• These mice are expensive/timeconsuming to make
• You must carefully observe the animals
for phenotype changes
– strange gait, spinning, too fat, too thin,
scaly skin…
Good Luck!