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Experimental Design and Paradigms

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cloning (aka somatic nuclear transfer): taking the nucleus from one cell ... Gurdon furthered the idea of cloning by culturing epithelial cells, and ... – PowerPoint PPT presentation

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Title: Experimental Design and Paradigms


1
Experimental Design and Paradigms
science is only as good as the controls you use
to show something 'true' 2 very different types
of controls, and both are essential negative
controls tell what is NOT the right answer
sometimes things are not related even when they
are found together the closer a negative
control is to an experiment, the better it
is positive controls tell that a technique
works and that negative results are not just
clumsy researchers if positive controls fail,
negative results mean absolutely nothing
2
Experimental Design and Paradigms
Several types of evidence are/were used for many
embryology studies 1) correlations two events
occur in sequence, suggests one causes
other weakest evidence-- could be coincidence
2) loss of function blockade of function using
antibodies or mutations has the problem of
specificity and non-specific effects 3) gain
of function artificially causing one action out
of sequence forces the second action-- often
involving gene expression
3
Experimental Design and Paradigms
Usually there are only a few approaches to a
problem that are reasonable 3 paradigms in
experimental embryology 1) what outside forces
control embryonic development 2) how do forces
in the embryo cause differentiation 3) how do
cells induce order into tissues and organs
2 major developmental strategies organisms use
1) lots of embryos few survive ie. fish spawning
2) few embryos, most survive ie. humans No
matter what strategy they use, they use generally
the same functional units-- most of the same
families of proteins are used by all animals key
differences are 1) when a particular protein
functions and 2) precisely how it induces its
effects-- small change big difference
4
the Politics of Science
just like others, scientists get very attached to
their ideas, and they can get violently
opposed to people who don't accept their
ideas one of the early questions was whether the
nucleus or cytoplasm controls
heredity embryologists wanted 3 things to
'prove' the genetic basis of development 1) how
do identical chromosomes produce different
variable cytoplasm? 2) do genes control early as
opposed to late stages of embryogenesis? 3) how
do genes regulate phenomena controlled by the
environment? ie. sex determination in
amphibians
5
Early Developmental Mutations
some scientists fought to unify genetics and
development brachyury one of the first
developmental mutants identified in mice
regulates development of the posterior part of
the embryo starts showing its effects very
early in the notochord fly wings were another
early target of genes and mutants- 100's today
brachyury in situ
bithorax mutation
6
Genome Equivalence
How do you prove that the genome of every cell is
the same? need to show totipotency totipotent
ability of a cell to carry out the development
of the organism therefore, need to be able to
1) enucleate host eggs without destroying them
2) method for isolating intact nuclei 3)
method for transferring isolated nuclei to a host
egg frog eggs proved very useful puncturing
with a sharp glass needle causes unfertilized
eggs to start developing meiotic spindle
can be removed
7
Genome Equivalence
Using a large glass needle, you can take a
nucleus from one cell and physically move it
to an enucleated egg this nucleus takes a small
amount of cytoplasm with it, but the egg is
huge-- almost all of the cytoplasm is from the
egg itself cloning (aka somatic nuclear
transfer) taking the nucleus from one cell
and putting it into an egg first accomplished
with frogs in 1952, 45 years before
mammals taking nuclei from blastulas was very
efficient-- many identical animals could be
produced taking nuclei from most later stages
was very inefficient suggested that somatic
nuclei lose their ability to direct developement
as they differentiate
8
Genome Equivalence
Gurdon furthered the idea of cloning by culturing
epithelial cells, and using those nuclei for
transplantation these fully differentiated cells
were able to take an egg up to neural tube
formation even though it didn't fully develop,
these nuclei could produce all of the cells in
the young tadpole blastomere nuclei were much
more able to be used in the cloning experiments
9
Genome Equivalence
1997, 'Dolly' was cloned in England-- first
mammal to ever be cloned from another adult
cell key step was to halt the cell cycle at G1
to collect the nuclei and oocytes had to be in
the second meiotic metaphase (ie. when normally
fertilized)
electroporation caused the cultured G1 cells
and oocytes to fuse and begin development only
1 of 434 attempts survived has been extended to
many species including cows, mice, cats, etc
10
Cloning Caveats
cloning is VERY inefficient compared to natural
fertilization/mating roughly 1 of embryos
develop normally (but they CAN do so) many
clones are defective in some way, with various
birth defects and developmental
abnormalities even though genetically
identical, many traits can vary between clones
ie. human twins, are individuals cloned sheep
from the same blastocyst can have different
sizes and temprament cloned cats (like calicos)
can be differently colored because their coats
are formed through a process of random X
chromosome inactivation
11
The Exception that proves the rule...
not all nuclei are created equal (only most of
them) immunoglobulins antibody genes in the
immune system made of 2 chains, heavy and
light, each composed of several segments
heavy chain larger of two chains, contains a
constant region recognized by immune system
cells light chain shorter, contains only the
antigen binding region
12
The Exception that proves the rule...
heavy and light chains are made from from two
different gene loci heavy chains have many
similar V and D regions, few J regions and 5
'constant' regions (changed during development
and selection) light chains have many V regions
and a few J regions during B cell (cells that
produce antibodies) maturation, DNA segments
between particular (random) regions are
permanently removed recombinases two particular
proteins in mammals that recombine DNA
mutations in either causes SIDS (severe immune
defficiency syndrome) 1000 different light
chain combinations are possible 10,000 different
heavy chain combinations can be made give
10,000,000 different antibody combinations
13
The Exception that proves the rule...
gives the immune system the ability to recognize
many different antigens so they can fight off
many diseases DNA regions at the junctions
between segments can also be slightly off
increases the variability even further AIDS is
such a problem because the cells that recognize
antibodies bound to their targets are T cells,
the very cells the AIDS virus targets
14
Differential Gene Expression
2 of 3 conditions are were met by the 1950's for
combining genetics and development-- now need
to show differential gene expression in
cells polytene chromosomes large, partially
replicated chromosomes found in fruit flies--
amplifies regions of chromosomes found in
several different cell types-- Beerman showed
that the DNA banding patterns were the same
except that different regions of the DNA
expanded in different cell types
15
Techniques of Genetic Analysis
Northern blot identifying RNA samples on a
gel 1) isolate RNA from several tissues 2)
separate RNAs on a denaturing agarose gel 3)
transfer the RNA from the gel to a nylon
filter blot transfer of biomolecule to a
filter 4) label a particular cDNA probe
complimentary to the RNA you're interested
in 5) block filter so it cannot bind anything
else 6) incubate labeled probe with filter
wash 7) develop the blot- identify size/tissue
of the RNA
16
Techniques of Genetic Analysis
northern blots can look at different tissues,
different treatments, or different ages blot
below is for a neural protein called
synaptobrevin in Xenopus it peaks at stages
35-45, the peak of synapse formation in frogs
17
Techniques of Genetic Analysis
Western Blot uses protein instead of
RNAs transfer to nitrocellulose filters using
electric current (40mV, 500mA) uses antibodies
to recognize proteins instead of a labeled
cDNA identifies the size of the protein instead
of the RNA Far-Western blot uses a normal
binding protein to stick to a natural partner
often uses a phosphorylated protein to identify
signaling molecules
18
Techniques of Genetic Analysis
Southern blot technique to analyze DNA cut
genomic DNA with restriction enzymes separate
electrophoretically like RNA blot like RNA onto
nylon block, then incubate with labeled
probe DNA from almost all tissues are
identical exception immunoglobulin
genes different T cells express 1 and only 1 Ig
RNA contains only some of the VDJ DNA
19
Techniques of Genetic Analysis
PCR polymerase chain reaction incredibly
sensitive technique toamplify a relatively small
known piece of DNA
20
Techniques of Genetic Analysis
PCR, antibodies, northern blots all identify
single genes at a time thousands of genes are
expressed at a time, so it would be more
efficient to study several thousand cDNAs at a
time genome projects allow you to do that--
sequences are now known must chemically
synthesize them, then put them on a surface,
often a glass slide or a blot of some
kind label 2 entire populations of cDNA with
different colors mix the two populations on 1
slide and see how much of each binds
21
Techniques of Genetic Analysis
green mRNA 1 red mRNA 2 yellow overlap
of red and green intensities of each color
represent the amount of that mRNA in the
inital samples each spot represents a
different mRNA this picture represents 800
northern blots!!!
22
in situ hybridization technique for localizing a
mRNA sequence within an embryo or tissue
section like a northern blot, starts with a
labeled probe that's complimentary to the
mRNA sequence embryo or tissue section is
incubated with the probe, excess is washed
away labeled probe is detected either
fluorescently, enzymatically, or autorad blue
color is caused by a reaction with alkaline
phosphatase, a common in situ detection method
23
antibodies can be used to stain tissue sections
and embryos as well immunohistochemistry use of
antibodies to localize proteins in a
tissue unfortunately, in situs and
immunohistochemistry give very descriptive
information, not causitive information microarray
s in particular are notoriously unreliable--
small differences get amplified, experiments
are qualitative instead of quantitative these
are still very useful experiments, but they are
not sufficient-- would prefer direct evidence
that a particular gene is responsible to get
that, you typically need to make some cell or
other express your gene of interest
24
tomato lectin staining in chick optic tectum
rat cardiac fibroblast
25
direct injection of mRNA is one way of putting a
protein in a cell only usable in relatively
large cells, like eggs very common for
studying early effects of genes can't use in
older animals-- cells won't stand being injected,
and cells will not express an injected mRNA
permanently but only for a time just like in
Acetabularia, the mRNAs eventually degrade and
run out
human egg being injected with a sperm for in
vitro fertilization
26
transfection incorporation of DNA into some
cell to express an mRNA, DNA must include a
promoter (region of DNA required to produce an
mRNA), a copy of the gene to make the protein
you want, and a termination signal vector piece
of DNA used to carry another piece of DNA and
usually controls it's expression
plasmid circular DNA from bacteria which
includes sequences for replication, selection,
(and often protein expression)
27
engineered viruses can also be used as a vector
for protein expression viruses include all of
the information needed to infect cells always
made so that they will not replicate in normal
cells require additional proteins provided by
special separate cell lines retrovirus RNA
virus that makes a DNA copy of itself and inserts
into the genome-- replicates only with the
cell- therefore labels all progeny most only
label actively dividing cells- most useful early
in development lentivirus variety of
retrovirus, includes the AIDS virus unlike most
retroviruses, lentiviruses can infect adult,
non-dividing cells-- very powerful tools because
they can be used at any stage of an organism's
life cycle
28
chimeric mice mice whose cells are derived from
two different sources embryonic stem cells
manipulated in vitro can be injected into
blastulas some of these cells become
incoporated into germ cells transgenic mouse
mouse containing extra DNA elements
intentionally incorporated into the mouse's
genome can include any kind of promoter
desired-- can have a protein expressed all the
time
29
(No Transcript)
30
knockout mouse specific type of transgenic mouse
where a desired gene is replaced with a
non-functional copy via homologous recombination
aka gene targeting or reverse genetics knockin
mouse specific type of transgenic mouse where a
target gene is replaced by some desired gene so
that it becomes expressed at the same times and
places as the target gene normally would
31
so far, the techniques we've discussed are good
for adding some new gene or replacing one gene
with another replacing a gene requires that you
know a lot about it, takes a lot of time, and
can only be done in very early embryos antisense
RNA direct injection of an RNA molecule
complimentary to a target mRNA-- antisense RNA
binds to the mRNA and prevents a ribosome from
acting upon it when it works it can mimic a
mutation in the target gene 'morpholino' RNA
chemically modified synthetic RNA-like molecule
which is not degraded by normal enzymes the way
RNA is-- much longer half-life in vivo
32
RNA interference (RNAi) targetted degradation of
a mRNA species by constructing a 22-23mer
synthetic double stranded RNA complimentary to
the mRNA recently discovered, RNAi is fast
becoming a technique of choice for seeing
effects of gene knockdowns in cells and
organisms dsRNA binds to several proteins (RNA
induced silencing complex) and specifically
cleaves targeted mRNA using the dicer enzyme
(RNAse) microRNA (miRNA) naturally occurring
small inhibitory RNA (siRNA) which are known
to block mRNA translation by not induce
degradation first identified in C elegans
development as a let-7 mutation causing larval
cells in adults, several hundred mRNAs are now
known in mammals degree of sequence identity
determines if a sequence will cause RNAi
33
NMDAR1 EF1a GluR1
normal
NMDAR1 EF1a GluR1
RNAi-1
NMDAR1 EF1a GluR1
RNAi-2
PCR to look at mRNA
controls
RNAi
NMDAR1
GluR1
schematic of RNAi
actin
western blots to look at protein
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