2 Methods of Study

8/6/2019 2 Methods of Study

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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 Amniocentesis- amniotic fluid is withdrawn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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2 Methods of Study