Class 10

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• Class 10

• Finishing up with Drosophila mosaic analysis.
• A little mouse interlude (transgenics, chimerics)
• The paper

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Techniques in Developmental Genetics Mosaic
Analysis
These cells are genetically mutant, and have a
visible marker that is linked to the gene of
interest (for example, a lack of pigment) The
color of the cell thus tells you the GENOTYPE of
the cell.but not its phenotype
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To answer the question of whether the gene of
interest acts cell autonomously or cell
non-autonomously, you ask two questions Are the
genetically mutant cells phenotypically
mutant? Are the genetically wild type cells
phenotypically wild type?
Note you cannot tell the difference between the
two kinds of autonomous proteins (RECEPTORS or
transcription factors)
Lets test your understanding.
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• 1. A mosaic fly has a patch of mutant cells (red
  cells) next to wild type (heterozygous) cells
  (white). Assume juxtacrine signaling.
• Which of the following results would indicate
  that the gene of interest
• acts cell autonomously?
• Cells in Columns 1 and 2 have a wild type
  phenotype. Cells in
• Columns 3,4,5 have a mutant phenotype.
• Cells in Columns 1, 2, and 3 have a wild type
  phenotype. Cells in
• Columns 4 and 5 have a mutant phenotype.
• c. Only cells in Column 1 have a wild type
  phenotype

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2. What will be the phenotype of cells if the
gene acts non-cell autonomously and the
arrangement of cells is as shown below? Assume
juxtacrine signaling again.
Genetically mutant
Genetically w.t.

• a. All cells in Columns 1, 2 and 3 will have a
  mutant phenotype
• All white cells will have a wild type phenotype
  all red cells will have a mutant phenotype
• All cells contacting the white cells will have a
  wild type phenotype
• All cells contacting the white cells will have a
  mutant phenotype

.5 pt
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Class 10 Developmental Genetics Mouse
Early embryogenesis
A 21-29
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Transgenic Mice Pronuclear Injection
Fertilized mouse egg
Transgene
Injection needle
1. Microinject the DNA into the pronucleus of a
fertilized mouse egg transgene is randomly
integrated
http//www.accessexcellence.org/AB/GG/transgenic.h
tml
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Transgenic Mice Pronuclear Injection
DNA microinjection into the male pronucleus of a
fertilized mouse egg
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Artificial regulation of expression-- express
gene in wrong place-- express mutant version of
a gene
What is done with transgenic mice?
Transgene DNA construct
Ask what happens to the mouse when this gene is
now expressed in the brain?
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Other Applications Produce large quantities of
human proteins

• 4. How do you know the milk will contain human
  AAT protein?
• AAT protein is produced in all cells of the sheep
• The human AAT gene is only present in the milk
  producing cells, thus only milk contains the AAT
• The transcription factors that activate
  production of AAT protein are only present in
  milk producing cells
• .5 pts

4.11
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What if you want to generate a line of mice that
is missing a specific gene? Do you mutagenize a
bunch of mice and screen through their progeny
for phenotypes that might indicate they are
missing that gene?
NOthat takes too long! You engineer the
permanent removal of a specific sequence of DNA
from the genome
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How? Use embryonic stem (ES) cells ES cell lines
are derived from cells of the early blastocyst
A 21-29
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Once you have a line of ES cells, you can
manipulate them
Introduce DNA into ES cells
Black mice
These ES cells now contain a new piece of DNA,
integrated into the genome in some way (well
discuss HOW soon)
Gilbert 4.19
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Use host mice that are white (you can use the
color later as a marker)
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Chimeric mice
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Will a chimeric mouse mated to another chimeric
mouse always generate some transgenic offspring?
These techniques, as described so far, have not
explained how you end up with a knockout
mouse Well discuss that next time!
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Persistence of Hunchback in the terminal region
of the Drosophila blastoderm embryo impairs
anterior developmentFlorence Janody, Joachim
Reischl and Nathalie DostatniDevelopment 127,
1573-1582 (2000)
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The GAL4 UAS system

• Your first task is to describe how they
  overexpressed hunchback.
• Helpful hints
• GCN4 is a yeast transcriptional activation domain
• When fused with GAL4, it appears to help
  stabilize the expression of whatever transcript
  GAL4 activates
• Thus, the transcripts overexpression wont be
  inhibited by other factors

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GAL4-UAS continued

• In this paper, GAL4 expression is driven by
• the nanos promoter (allows for expression in the
  MOTHERdoesnt matter that its a posteriorly
  located mRNA normally)
• AND the bicoid 3 UTR, which targets the message
  to the anterior only.
• Because GAL4 is expressed in the anterior, the
  protein is only made in the anterior. The GAL4
  protein then binds to the UAS sequence upstream
  of hunchback cDNA
• Hb now overexpressed in anterior

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How do bicoid hunchback nanos
torso function in the embryo? What would be
the phenotypes of lf mutations in these genes?
Hunchback early zygotic transcription
Torso activated at termini
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This figure shows where GAL4 (and thus hb) are
expressed in GAL4 UAS flies
In A. Lac Z is driven by expression of GAL4
protein in anterior (shows where Hb will be
expressed)
persistent maternal hb
These embryos are all at a mid-cellularization
stage
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All embryos from bicoid -/ mothers (whether the
embryos are / or -/) receive only 1 copy of
maternally supplied bicoid These embryos, if they
also have extra Hb expression, experience even
higher lethality than Hb embryos alone
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Fig. 7. Repression of hb by Torso is not
sufficient for cnc, wg and hh early anterior
expression. In situ hybridisation on blastoderm
hb -zygotic embryos with cnc, wg and hh
probes. hb -embryos were identified through their
ftz expression pattern detected with a
simultaneous staining using a ftz probe. Embryos
were from torso (A-C) and wild-type (D-F)
females.
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C. elegans mosaic recap, if time

• ICQ from class 8

• 3. You have made a stock of worms homozygous for
  a strong loss-of-function lin-3 mutation and
  carrying a small free duplication that includes a
  lin-3() gene. Most of these animals are
  phenotypically wild type. From this strain, you
  isolate an animal that is genetically mosaic for
  lin-3 function, as the result of losing the free
  duplication in the AB cell with retention in P1
  during the first mitosis in the embryo. What
  will be the phenotype of this animal with regard
  to vulval development?
• Vul
• Muv
• Wild type