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exploration of living cells
using extremely minuteinstruments as
microneedles, microhooks,
& micropipettes.
Specimens are positionedwithin an operating
chamber on the stage of
the compound microscope
by mechanicalmicromanipulators
capable of achieving
controlled movements in
various planes.
WHOLE
CELL/ORGANISM
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Procedure for transplanting blastula nuclei into activated
enucleated Rana pipiens eggs. The relative dimensions ofthe meiotic spindle have been exaggerated to show the
technique. The handsome prince Freddy was derived in
this way. The vitelline envelope is the extracellular matrix
surrounding the egg.
Freddy
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A clone of Xenopus
laevis frogs. The nuclei for all
the members of this clonecame from a single
individuala female tailbud-
stage tadpole whose parents
(upper panel) were both
marked by albino genes. Thenuclei (containing these
defective pigmentation
genes) were transferred into
activated enucleated eggsfrom a wild-type female
(upper panel). The resulting
frogs were all female and
albino.
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2. Cell-labeling/ tracing methods
Carbon particles and nontoxic fluorescent dyes are
taken up superficially by cells
Introducing intracellularly enzymes with expected
histologic outcomes, e.g. horseradish peroxidase
(HRP)
Retroviruses
containingreporter genes
(e.g. human
alkaline
phosphatase
hPLAP, E. colibeta galactosi-
dase, the jellyfish green fluorescent protein GFP).
The presence of such indicator substances allows
tracing of path of cells during morphogenesis.
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3. Use of Mutants- use of mutant forms and
genetic markers (e.g. T mutations in mice) to
determine genetic pathways controllingcellular interactions in the early embryo.
4. Inhibitory Agents and
Teratogens- cytoxic
substances that causedamage to embryonic
cells allow detection
of developmental stage
and appearance of
affected tissues/organs
Thalidomide baby
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5. Amniocentesis- amniotic fluid is withdrawn
and examined for possible chromosomal
abnormalities or other genetic defects
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6. Mathematical Models: When 2 homogeneously
distributed solutions
interact they produce
stable patterns during
morphogenesis.
These patterns wouldrepresent regional
differences in the
concentrations of the two
substances.
Their interactions wouldproduce an ordered
structure out of random
chaos (Alan Turing,1952).
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Isometric growth- the shape is preserved because all
components grow at the same rate
Allometric growth (or allometry)- different growthrates of parts within the same organism because not
all parts of the body grow at the same rate
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In situ hybridization performed on a whole chick
embryo that have been fixed without beingsectioned. The probe used recognizes the mRNA
encoding Pax6 in the chick embryo. This probe is
labeled not with a radioactive isotope, but with a
modified UTP. To create this probe, a region of the
cloned Pax6 gene was transcribed into mRNA,
containing UTP conjugated with digoxigenin. Thisdoes not interfere with the coding properties of
the resulting mRNA, but does make it recognizablydifferent from any other RNA in the cell.
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Identifying the
Genes for Human
Developmental
Anomalies
There is no single
pathway to
success, but the
key step is to arrive
at a plausible
candidate gene,
which can then betested for mutations
in affected people.
Note the interplay
between clinical
work, laboratory
benchwork and
computer analysis.
Database searching
is becoming more
and more crucial as
information from
genome projects
accumulates.
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For example, a gene fragment is
absent in chromosome 11 of
individuals affected with
aniridia (absence of eyes/nose). Northern blot analysis & in situ
hybridization showed that this
fragment contains the Pax6
gene, expressed in the brain
and eye.
1. POSITIONAL
GENE
CLONING-
finding theregion in a
genome where
a particular
mutant gene is
thought toreside.
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Human
Waardenburg
syndrome2
(deafness, eyeabnormalities, white
forelock) has
equivalent
mutations in the
mice gene for
microphthalmia.
The mouse mi gene
was cloned, & used
to make a probe
which looked for its
human homologue,the MITF locus
which mapped to
the exact region as
the Waardenburg
syndrome2.
2. CANDIDATE GENE
MAPPING- correlation
between the genetic
mapping of a particularsyndrome and themapping of a gene.
2. CANDIDATE GENE
MAPPING- correlation
between the genetic
mapping of a particularsyndrome and themapping of a gene.
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3. REPORTER GENE - expression is easy to detect;
regulated by DNA of interest
LacZ (Beta-Galactosidase) cleaves substrateto form blue color
GFPGreen FluorescentProtein is green
under fluorescent light
Reporter Gene (GFP, LacZ)Regulatory region
Visualize Reporter Gene
LacZ & Reporter Gene GFP
Recom-binant
DNA
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4. MICROARRAY TECHNIQUE. Target mRNAs are prepared by isolating
mRNAs from two cell types being compared (two different cell types or the
same cell type at different times), making cDNAs from them. In this case,
cDNAs from cell type 1 are labeled with a green dye, while cDNAs from cell
type 2 are tagged with a red dye. Microarray probes are made by taking anumber of different cDNAs made from the mRNA of one cell type and
adhering them to a glass microscope slide. The probes and the target are
hybridized together. If the mRNA in a probe is abundant in cell type 1, the
signal is green. If the mRNA is abundant in cell type 2, the signal is red. If
the mRNA is present in both cell types, the signal is yellow. The bottom
shows a portion of such a microarray.
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5. MACROARRAY ANALYSIS of those genes whose expression in the
early Xenopus embryo is caused by the activin-like protein Nodal-
related 1 (Xnr1). (A) Creation of targets for macroarray analysis.(B) In the macroarrays, certain radioactive spots (representing
hybridizations) were seen in samples from the Xnr1-stimulated cells,
but not in the control cells. These spots represent the genes activated
by Xnr1; the insert shows one of these, the chordin gene. Most of the
thousands of genes observed were not activated. The DNA of the
hybridized spots can be sequenced and identified.
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Integratedgenetic,
physical
and
sequence
map
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www.mcw.edu/bioethics/
www.nih.gov/sigs/bioethics/index.html
http://www.dnalc.org/resources/animations/cloning101.
html
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://www.dnalc.org/resources/animations/pcr.html
http://www.dnalc.org/resources/animations/dnaarray.ht
mlhttp://www.bio.davidson.edu/courses/genomics/chip/
chip.htmlhttp://learn.genetics.utah.edu/content/labs/microarray/
http://learn.genetics.utah.edu/content/labs/extraction/
www.georgetown.edu/research/nrcbl/nirehg/index.html
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