Transcript Using Transgenic Technology to Characterize Regulatory Regions

PowerPoint to accompany
Genetics: From Genes to Genomes
Fourth Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver
Reference
E
Prepared by Malcolm Schug
University of North Carolina Greensboro
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Mus musculus:
Genetic Portrait of the House Mouse
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Fig. E.1
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Outline of Reference E
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Overview of Mus musculus in laboratory
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Mouse genome
Mouse life cycle
Transgenic protocols
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Uses of transgenic technology to determine
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Addition of genes by nuclear injection
Removal of genes by targeted mutagenesis
Function of gene products
Characterizing regulatory regions
Links between mutant phenotypes and transcription
units
Creating a mouse model for human disease
The Hox genes: a comprehensive example
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The Mouse Genome
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Similar to humans
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Contains about 3 billion nucleotides
Homologues for every gene in humans
Differences due to species-specific additions to gene families
19 autosomes and 2 sex chromosomes
No evidence of banding similarities with humans in
karyotypes
Conserved synteny
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Genes closely linked in mouse are also closely linked in humans
Average size of conserved syntenic regions: 17.6 Mb
Since common ancestry, human and mouse genomes have broken
apart and rearranged ~ 170 times
May identify a gene in one species and locate it in other species
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Table E.1 Comparison of Mice and Humans
Trait
Mice
Humans
Average weight
30g
77,000 g (170 lb)
Average length
10 cm (without tail)
175 cm
Genome size
3,000,000,000 bp
3,000,000,000 bp
Haploid gene
number
25,000
25,000
Number of
chromosomes
19 autosomes + X and Y
22 autosomes + X and Y
Gestation period
3 weeks
38 weeks
Age at puberty
5-6 weeks
624-728 weeks
Estrus cycle
4 days
28 days
Life span
2 years
78 years
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Conserved
Synteny
Fig. E.3
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Life Cycle
Fig. E.4
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Germ Cell Development
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Males
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Sperm
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Fig. 4.21
At puberty, large number of haploid
gametes produced for rest of life
Spermatogenesis
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Germ Cell Development
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Female
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Born with all gametes they will ever have
~ 50,000 eggs, or oocytes
Oogenesis begins in ovaries of fetus
Oogonia enter meiosis but stop at diplotene in first
prophase
Estrus cycle begins at puberty
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4 days in mice
8-10 primary oocytes complete first meiotic division
Secondary oocyte stops at metaphase are released from ovary
(ovulation)
Oocyte passed into oviduct and is receptive to fertilization
(estrus)
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Figure 4.18
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Fertilization
Fig. E.5
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Early Embryo Development
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Fig. E.6
Preimplantation starts at
conception
22 hrs sperm head
expands into pronucleus
Next 60 hours – embryo
divides four times equally
(totipotent) – cleavage
stage
Quadruplets from single
fertilized egg
Chimeras
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Events Restricting the Developmental Potency
of Individual Cells Occur Near the End of the
Preimplantation Stage
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16-cell embryo
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First differentiation events occur
Trophectoderm layer – cells on outside of embryo
Inner cell mass – cells on inside of embryo
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Fetus is derived from ICM
Blastocyte forms
After implantation, placenta develops, embryo
grows, and tissues and organs emerge
Birth at 21 days after conception
Suckling period lasts 18-25 days
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Two Powerful Transgenic
Techniques
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Addition of genes by nuclear injection
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Foreign DNA injected into pronucleus of fertilized egg
Place injected one-cell embryo back into oviduct
25-50% of time DNA integrates at random into chromosome
Targeted mutagenesis
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Embryonic stem lines divide without differentiating
Cultures of thousands; some of which will be injected back into
blastocoele
Integrated into germ line
Homologous recombination may occur after transfection
Knockout constructs have nonfunctional gene that can be
exchanged for mouse gene by homologous recombination
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How
Transgenic
Mice are
Created
Fig. E.7
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Knocking Out a Gene in ES Cells
Figure E.8
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Using Transgenic
Tools
Determine gene
function
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Combining mouse gene
with regulatory regions
from other mouse gene
Transgene expressed:
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Fig. E.9
At higher than normal
level
In an alternative tissue
At alternative
developmental stage
Helps determine
function of wild-type
gene
SRY locus responsible
for production of
maleness
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Application of Transgenic
Technology
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Fig. E.10
Transgenic
expression of myc
gene provides
information on
gene’s role in tumor
formation
(a) structure of gene
(b) northern blot
analysis
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Using Transgenic Technology to
Characterize Regulatory Regions
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Fig. E.11
DNA construct
containing mouse
regulatory region of
interest is attached
to E. coli reporter
gene
Function ascertained
by b-gal expression
in transgene fetus
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Fig. E.12
Use of transgene
technology to map
cis-acting regulatory
region of Tcp 10bt gene
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Fig. E.13
Transgenic technology to
identify locus responsible
for mutant phenotype
Dominant deletion
mutation at T locus
causes short tail
Transgene mouse with
pme75 transgene mated
to mutant
Normal phenotype
demonstrates deletion of
pme75 is cause of short
tail
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Targeted Mutagenesis to Create a Mouse Model
for Human Disease
Fig. E.14 a-c
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Fig. E.14 d,e
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Fig. E.14 f
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Inheritance Pattern
Fig. E.14g-h
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The Hox Genes: A Comprehensive
Example
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Homeotic selector genes (Hox)
4 gene clusters with 9-11 genes each
 Control development of body segments
 Used Hox genes of Drosophila as probes in
cross-hybridization studies
 In flies, homeotic genes active in the discrete
segments that define body plan, where they
determine proper differentiation of tissues
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Mouse Hox Gene Superfamily Contains Multiple
Homologs of Each Member of the Drosophila Homeotic
Selector Gene Family
Fig. E.15
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Analysis of Expression Patterns in Developing
Embryos Provides Clue to Time and Location of
Gene Action
Spatial extent of expression in some Hox genes along developing spine
Fig. E.16
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Validating the Hypothesis that Expression of the 5
Gene in a Hox Cluster is Epistatic to Expression of
the More 3 Genes
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Fig. E.17
Transgenic construct
misexpresses HoxD4 in
more anterior region
where HoxA1 is
normally located
Skeleton of wild-type
(left) and transgenic
animal (right)
Enlargement of
cervical regions from
skeletons shown below
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Knockout Studies Confirmed
Predictions of Aberrant Phenotypes
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5 epistasis hypothesis leads to prediction
that aberrant phenotypes will arise when
different Hox genes are knocked out by
homologous recombination
Data using knockouts support this
hypothesis
e.g., knockout of HoxB4 produces a partial
homeotic transformation of second cervical
vertebra from axis to atlas
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Fig. E.16
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