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Genomic equivalence
• Various models were used to explain how
cell division leads to differentiation
• Arguments over the nucleus vs. cytoplasm
as source of developmental “instructions”
• Autonomous specification led to the germplasm therory of Weissman (1883)
• Nuclear determinants are segregated by
cell division resulting in unequal nuclei
The Weissman Theory
Mosaic development/Autonomous specification
The experiments of Roux (1888) seemed to support this model
of differentiation (see fig 3.14 and pages 58-59 of text)
Regulative development/conditional specification
Fig 3.15
Page 59
Gilbert
This and other experiments suggested that the nuclei of cleavage
products remain equivalent and capable of forming a whole embryo
Nuclear transplants in Frogs
Nuclei of donor
cells remain
totipotent
during
development
Up to a
point…...
The final proof of
the principle of
genomic equivalence
Dolly the Lamb and
Cumulina the mouse
and their offspring
Dolly:
Wilmut, et. al. (1997) Viable offspring
From adult and fetal mammalian cells
Nature 343:657:81-814
Cumulina:
Wakayama, et. al. (1998) Full term
Development of mice from enucleated
Oocytes injected with cumulus cell nuclei
Nature 394:369-374
Cloning is laborious and inefficient
From Wakayama and Yanagimachi (1999)
Only approximately 1% of oocytes
injected with cumulus (somatic) cell nuclei
develop to term--WHY??
A lot happens to a nucleus of a fertilized egg
Key problems are nuclear reprogramming and cell cycle coordination
Cell cycle coordination
• Donor nucleus must adopt the cell cycle
parameters of a zygote
• Donor nucleus needs to be in G0 or G1
phases of the cell cycle (quiescent-starved)
• (S or G2 phases:BAD) (G2/M phase:BAD)
• Therefore initial cell cycles can be
controlled by the recipient (oocyte)
cytoplasm (as normally occurs)
Zygotic vs. Somatic cell cycles
MPF is maternally derived and it determines the initial cell cycles
Somatic cell cycles have growth phases
Nuclear reprogramming
• Somatic cells are transcriptionally active
whereas a fertilized egg is NOT
• All of the epigenetic changes that created
the somatic cells need to be “erased” so that
the state of the chromatin of the
transplanted nucleus is more “neutral”
• Epigenetic changes which normally occur
during embryogenesis start anew
Changes in chromatin structure
• 75% of pre-existing protein is lost from the
somatic nucleus following transplantation
• Involves changes in the following:
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–
–
–
–
Histone components and modifications
Chromatin remodeling complexes
DNA methylation status
Transcription factors
Repressive chromatin (heterochromatin) proteins
Cloning and development
• Events following nuclear transplantation
mimic normal events which initiate
embryonic development
• Genomes are equivalent
• Differences in “use” of the genome occurs
through epigenetic regulation
Exceptions to genomic equivalence
• B-lymphocytes and T-lymphocytes
• Both cell types utilize somatic DNA
rearrangement to create antigen receptors
• Results in PERMANENT LOSS of DNA
from the genome
• Lymphocytes utilize multiple strategies to
generate antigen receptor diversity. Many
of these make permanent changes to DNA
Antibody gene assembly
TdT
N-region addition
RAGs
DNA repair
Somatic
hypermutation
DNA altering processes in
antigen receptor generation
• Somatic recombination (V-D-J)
– Involves specific recombination enzymes (RAG1
and RAG2)
– Involves DNA repair machinery (DNA-PK, Ligase)
• N-region addition (TdT enzyme)
• Somatic hypermutation (linked to transcription)
• Class switch recombination (unknown)
Techniques to detect gene
expression (mRNA)
•
•
•
•
•
Northern blot
RNase protection assay
Reverse transcriptase-mediated PCR
In situ hybridization
Subtractive hybridization
– Allows cloning of unknown genes that are
differentially expressed
Northern blotting:
Separates mRNA by size
via denaturing agarose gel
electrophoresis. The
separated mRNAs are
transferred to a nylon
membrane. A radioactive
DNA probe that is
complimentary to the
mRNA is used to detect
the message of interest by
hybridization.
In situ hybridization
Tissue sections are prepared
and cleared of DNA. mRNA
is denatured and the labeled probe
is used to detect the position
within the tissue of the mRNA
Reverse transcriptase mediated PCR
(RT-PCR)
RNase protection assay
• Prepare a 32P-labeled probe and hybridize
to total RNA prepared from cells of interest
• Digest for a short time (30 min) with RNase
(A + T1)
• Presence of mRNA complementary to the
probe will protect it from digestion.
• Purify undigested RNA complexes
• Denature and run on acrylamide gel
Quantification of RNAse protection data
Subtractive mRNA screen strategies
• Subtractive hybridization
• Differential display
• Representational difference analysis (RDA)
• Gene chip analyses
Conditions which make for a
succesful subtraction
• Target mRNA should be abundant
• Should be at least a 3-fold difference in
expression level between the 2 populations
• Target gene should be differentially
transcriptionally regulated
• Many artifacts results from this kind of
procedure. Need to carefully control it
Discussion of RDA
• Reference: Pastorian, et. al. (2000) Anal.
Biochem. 283:89-98 (will post to website).
This is an “improved” RDA method.
• Pools of RNA from different cell populations
are reverse-transcribed into DNA
• DNA is restriction enzyme digested into small
pieces for easy amplification in PCR
RDA continued
• The “tester” mRNA pool contains the target
mRNA, the “driver” pool does not. The
driver is used to “subtract” mRNAs present
in both cell types
• Tester pool is ligated to a linker (used for
PCR). The “driver” pool has no linker
• Driver hybrids do not amplify in PCR,
Tester/driver hybrids amplify linearly
Tester/tester hybrids amplify exponentially
Next lecture:techniques used to study
the role of genes in develpoment
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Random genetics followed by screening
Targeted mutagenesis (gene knockout)
Transgenic animal models
Dominant negative mutant molecules
Antisense RNA interference
RNA interference (c. elegans-website 4.8)