Download BIO 402/502 Advanced Cell & Developmental Biology

The Code For Life
Organism
.
Tissues
Organ
System
Cell
Nucleus
The Code For Life
Chromosome
Big nose
Brown eyes
Nucleus
Straight hair
Genes
Structural Biology
Medicine and Biology at the Atomic Scale
Organ  Tissue  Cell  Molecule  Atoms
 A cell is an organization of millions of molecules
 Proper communication between these molecules is
essential to the normal functioning of the cell
 To understand communication between molecules:
*determine the arrangement of the atoms*
Advanced Cell &
Developmental Biology
Gene, Recombinant DNA
& Cloning Analysis
Restriction Enzymes
• Restriction enzymes are DNases
(nucleases) found in bacteria that recognize
specific DNA sequences as 4mers,6mers or
8mers and make double stranded breaks in
DNA .
• This enables cutting of genome in specific
ways to generate restriction site maps and
the development of approaches for pasting
pieces of DNA together in specific ways.
A
Separation
of EcoR1
B
segments
C
on an
D ,E agarose gel
F
DNA Hybridization
• DNA hybridization is the process whereby complementary strand of DNA anneals
(to form a double helix) with the single stranded DNA
• Hybridization can be measured by labeling the “complementary strand” either with
32P nucleotides or fluorescent probes .
• There is also DNA-RNA hybridization
Southern Blotting
• Southern Blotting enables identification of specific DNA sequences (gene
fragments) from among the total sequence of DNA
Cut DNA with restriction
enzymes
Separate fragments on
agarose or acrylamide
gels
Transfer the separated
DNA from gel on to
nitrocellulose paper
Hybridize with a labeled DNA or RNA of
interest ( e.g., 32P labeled DNA) followed
by autoradiography or phosphoimaging
for detection
Northern Blotting
•
Northern Blotting is where RNA is blotted and then probed labeled DNA (cDNA)
synthesized from the mRNA isolated from the cell
• Enables identification and quantification of specific mRNAs from among the vast
population of RNAs in the cell
DNA cloning
• DNA cloning enables specific
pieces of genome to be inserted
into bacteria as plasmid or
phage lambda vectors and
grown in large quantity.
• The first step is to generate a
library of bacteria with inserted
DNA fragments. This could
either be a genomic(DNA)or a
cDNA (mRNA) library
Replica plating and in situ hybridization
• Techniques used to identify a bacterial colony that contains the gene (DNA sequence)
of interest. The isolated colony can be grown up in large quantities.
CsCl centrifugation for separation of plamid DNA
from chromosomal DNA
Replica plating and in situ hybridization
cDNA libraries
• They are generated to isolate
particular genes of interest or to
identify a gene based on the protein
expression of that gene cloned in
the bacterial cell
• The latter procedure is called
“reverse genetics” whereby the
protein product is used to identify
the gene followed by DNA
sequencing
DNA sequencing
•
Sanger’s dideoxy method
DNA to be sequenced is
mixed with each of 4
ddNTPS (chain
terminators) in separate
reactions for DNA
synthesis and later
separation of the products
by electrophoresis
•
Can now be done
automatically via
sequencing machines that
work with different
flurochromes attached to
each of dideoxy
nucleotides
•
To determine the sequence
of a gene of many
kilobases overlapping
DNA fragments of 400800 bp must be sequenced
Protein expression vectors
• These are specially designed plasmid
vectors for fusion protein expression
to isolate large quantities of protein of
interest for antibody production or
other studies of purified protein.
• The proteins are produced as fusion
proteins of the cDNA gene coding
sequence ligated to a protein
expression marker or reporter protein
e.g. beta-galactosidase
• They can also be used as a major tool
in cell biology to study the expression
of proteins in cells following DNA
transfection
DNA transfection and Polymerase chain reaction (PCR)
• DNA transfection is used to
Polymerase chain reaction (PCR)
track the properties of individual proteins
in a cell
Is used as an alternative to cloning
for purifying a particular DNA (gene
sequence
Construct a plasmid expression system
that contains the protein of interest fused
with a reporter gene such as a betagalactosidase or a short peptide sequence
such as HA 9 mer peptide or FLAG
epitope for antibody localization with
anti HA or anti FLAG or fluorescent
localization in living cells with GFPconstructs (GFP-actin)
It enables the production of microgram
quantities of the DNA sequence of
interest in the test tube
Provides an alternative for preparing DNA
probes to screen genomic or cDNA library
for clones encoding a protein of interest
DNA Microarrays and chips
• Enable via fluorescence in situ hybridization (FISH) to measure expression
of 1000’s of genes on each array/ chip.
Actual chip size
Yeast genome microarray: The array is hybridized to cDNA
labeled with a green fluorescent dye prepared from cells grown in
glucose and with red labeled cDNA from cells grown in ethanol.
Spots were detected with a scanning confocal microscope
Antibody production
• Polyclonal antibodies are
generated by injecting
antigen into an animal and
purifying the antibody
titer from blood
• Monoclonal antibody
technique enables to obtain
a single clone of cells that
recognizes one epitope
( usually ~ 9 a.a.) of the
total protein
Monoclonal antibody production
Genetic Engineering
• Introduction of exogenous genes ( mutant or
normal) in to normal cells or organisms to study
gene expression
• Used to study the role of the protein coded by the
gene in the cell/organism function or for
engineering gene expression for improving food
production or reducing the destrcutive damage of
human diseases
Site Directed Mutagenesis
• Alterations in nucleotides (substitutions or
deletions) in vitro at known (directed) sites to
create “mutant genes”
• These mutant genes can be transfected into cells as
previously discussed and enables study of gene
function at the individual cell level. The
transfected genes are also called “transgenes”
Production of transgenic mouse
Inject mutant gene
in to one of the
pronuclei of the
fertilized mouse
oocyte
Transfer oocyte to
surrogate mother. 1030% of offspring
contain the transgene
in equal amounts in
all tissues
Gene Knockout or “replacement”
• Form of trangenics
• Occurs following homologous recombination of the transgene at the site of the
endogenous gene
• Occurs readily in yeast cells but in mammalian cells the rate of recombination is very
slow and hence a double selection marker approach is adopted where the first marker
e.g. neomycin resistance selects for all cells with homologous recombination while
the second marker allows growth of only those cells that carried out homologous
recombination
Knockout protocol
ES cells are isolated
from the inner
blastocyst and
culture
ES cells are
tranfected with
the gene of
interest
ES cells successfully
transfected via homologous
recombination are selected
and grown in culture and
injected into a host
blastocyst. Chimeras
develop which contain ES
cells from both the
transfected and the host
cells.
Enables
direct study of
gene function
in an intact
organism
Gene Replacement/therapy
• Replace an abnormal
gene with a normal one
at a very early stage of
development
• It has the potential for
curing or alleviating the
symptoms of a wide
variety of human
diseases, e.g.,Parkinson’s
disease
Procedure for gene replacement
How Ian Wilmut Made Dolly 1
Making Quiescent Cells
Mammary gland cells
Finn Dorset ewe
3.5 months pregnant
Culture mammary cells
Starve cells
Harvest quiescent
cells
How Ian Wilmut Made Dolly 2
Collecting The Donor Nucleus
Glass pipette
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 2
Collecting The Donor Nucleus
Glass pipette
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 3
Egg Preparation
Egg
Scottish Blackfaced
ewe egg donor
An egg is collected then
placed into a dish where it
can be manipulated
How Ian Wilmut Made Dolly 3
Egg Preparation
Glass pipette
Egg
Chromosomes
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 3
Egg Preparation
Chromosomes
Glass pipette
Egg
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 4
Inserting The Donor Nucleus
Glass pipette
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 4
Inserting The Donor Nucleus
Glass pipette
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 4
Inserting The Donor Nucleus
Suction
Suction
Pipette
How Ian Wilmut Made Dolly 5
Initiating Development
How Ian Wilmut Made Dolly 5
Zygote
Initiating Development
How Ian Wilmut Made Dolly 5
Cleavage
Initiating Development
How Ian Wilmut Made Dolly 5
Cleavage
Initiating Development
How Ian Wilmut Made Dolly 5
Cleavage
Initiating Development
How Ian Wilmut Made Dolly 5
Cleavage
Initiating Development
How Ian Wilmut Made Dolly 5
Morula
Initiating Development
How Ian Wilmut Made Dolly 6
Development
Morula
Scottish Blackfaced
ewe surrogate
mother
Finn Dorset lamb
Dolly