LATg Training Course

igocheddarBiotechnology

Dec 14, 2012 (4 years and 11 months ago)

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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


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
CGAAG
G
AATTC
CGTAG
TGATT
G
AATTC
TTAAC
GCTTC
CTTAAG
GCATC
ACTAA
CTTAAG
AATTG
+ Eco R1
CGAA
G

A
ATTC
CGTAG
TGATT
G

A
ATTC
TTAAC
GCTT
C
TTAA


G
GCATC
ACTAA
C
TTAA

G
AATTG
+ ligase (another enzyme that attaches the m/c)


CGAA
G

AATTC
TTAAC

GCTT
CTTAA
G
AATTG

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/time
-
consuming to make


You must carefully observe the animals
for phenotype changes


strange gait, spinning, too fat, too thin,
scaly skin…

Good Luck!