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Genome Engineering

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Genome Engineering. Goal: Manipulate pieces of DNA to build recombinant ... Zebrafish -REMI -Transposase systems working and gaining popularity. Mouse ... – PowerPoint PPT presentation

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Title: Genome Engineering


1
Genome Engineering
2
Molecular Biology Revolution
Goal Manipulate pieces of DNA to build
recombinant constructs with a defined composition
and sequence
Approach Steal enzymes, vectors and other
tricks from simpler systems Tools Enzymes
restriction enzymes, DNA ligases, RNA and DNA
polymerases, etc. Vectors Plasmids,
bacteriophage (lambda, P1), BACs, YACs Hosts
bacteriophage and bacteria (sometimes yeast)
In order to create -Recombinant plasmids for
cloning and study of foreign DNA, protein
expression, site-directed mutagenesis, etc. etc.
etc. -cDNA and genomic libraries -whole genome
chips etc., etc., etc.
3
Genome Engineering Revolution
Goals Manipulate the genome of higher
organisms, including humans, to facilitate more
sophisticated genetics and therapies.
Approach Use Molecular Biology to build DNA of
interest and use new tricks for introducing and
manipulating foreign DNA in the
genome. Requirements Genetic system Ways to get
DNA -into cells -into genome -into
germline Tools Enzymes integrase/recombinase,
transposase Vectors transposons, viruses
In order to -Mutate genes of interest (globally
or just in some cells) -Identify new genes -Study
regulatory DNA and gene expression -Ectopically
express foreign or modified genes
4
Getting DNA Into a Cell (That Can Form a Germ
Cell)
1) Injection -inject into syncytial gonad in C.
elegans -inject into the syncytial embryo in
Drosophila -inject into the nucleus of mouse
oocyte
2) Infection Use viruses to infect cells or
embryos -can be used for transient gene
delivery -can go germline if use ES cells or
infect right cells of embryo -used for
insertional mutagenesis in mouse and fish
3) Transfection Cells will take up DNA under
correct treatment e.g. -when DNA is in calcium
phosphate ppt. -when DNA is incorporated in
liposomes (lipofection) -when cells are exposed
to electric field (electroporation)
5
Getting DNA Into a Genome
1) Random Integration -Linearized DNA will often
incorporate randomly into the genome -Often
incorporates in multiple copies -Can be enhanced
by using Restriction Enzyme Mediated Integration
(REMI) Supply cells with same RE used to
linearize DNA
2) Transposon or Retroviral Insertion -Place DNA
of interest in transposon or retroviral
backbone -Supply DNA Transposase OR Produce
packaged virus and infect cells -Usually get
single integration -Integration may not be
random, depending on insertion preferences of
system
6
Transposons
-Mobile genetic elements that can jump around
genome -DNA transposons utilize Cut and paste
mechanism for jumping (remain relatively low copy
) -Retrotransposons utilize RNA intermediate and
copy and paste tranposition (increases copy
--LINE transposon alone makes up 21 of human
genome!) -Transposons can be separated from
transposase so transposition is controlled
independently -Transposons can be vehicles for
transgene delivery and mobilization
7
Requirements -DNA of interest cloned into vector
between transposon inverted repeats -Dominant
selectable marker to screen for
transformants -Separate source of
transposase Inject vector transposase and
construct will hop off plasmid and into genome
8
Viruses as Vectors for Genome Engineering
Advantages -Can act as vehicle to deliver DNA
into cell AND into genome -Can be modified in
similar ways as transposons Also useful for
transient delivery of DNA into cells -Express
gene/construct of interest during development by
exposing embryo to virus -Gene therapy by
expressing gene/construct of interest transiently
in human cells Disadvantages Cant always supply
virus to germ cells More difficult to control
than transposons (cant jump them around)
9
Getting DNA Into a Genome
1) Random Integration -Linearized DNA will often
incorporate randomly into the genome -Often
incorporates in multiple copies -Can be enhanced
by using Restriction Enzyme Mediated Integration
(REMI) Supply cells with same RE used to
linearize DNA
2) Transposon or Retroviral Insertion -Place DNA
of interest in transposon or retroviral
backbone -Supply DNA Transposase OR Produce
packaged virus and infect cells -Usually get
single integration -Integration may not be
random, depending on insertion preferences of
system
3) Integration into a Defined Landing
Site -Combine DNA of interest with integration
sequence (e.g. from bacteriophage) -Use host
whose genome has already been modified to contain
landing site -Supply DNA Integrase
10
-Normally used by bacteriophage for lysogenic
cycle -Classic studies with lambda
phage -Promote SITE-SPECIFIC integration -Many
systems have -Single Integrase with no
additional factors needed -Works in vitro and in
other hosts (flies, mice) -Some are irreversible
(and therefore VERY efficient) forward rxn
requires only integrase reverse rxn requires
integrase excisionase -Basis for Gateway
cloning system -CRE/lox system is another example
from bacteriophage P1 -FLP/FRT system is related
system from Yeast
11
Integrases/Recombinases
Advantages -Specific insertion site -Very
efficient (with irreversible integrases) -easier
transformation and larger constructs Disadvantage
s -Need target sequence in genome -Specific
insertion site -cant do random
integrations/screens -Cant mobilize after
integration
12
Site-specific Recombinases Precision tools for
genome modification
FLP Recombinase and FLP Recombinase Targets (FRT)
in Drosophila Stolen from yeast by Golic and
Lindquist, 1989 Cre Recombinase and loxP target
sites in Mouse Stolen from bacterophage P1 by
Sauer and Henderson, 1988
Garcia-Otin and Guillou, 2006
13
Getting DNA Into a Genome
1) Random Integration -Linearized DNA will often
incorporate randomly into the genome -Often
incorporates in multiple copies -Can be enhanced
by using Restriction Enzyme Mediated Integration
(REMI) Supply cells with same RE used to
linearize DNA
2) Transposon or Retroviral Insertion -Place DNA
of interest in transposon or retroviral
backbone -Supply DNA Transposase OR Produce
packaged virus and infect cells -Usually get
single integration -Integration may not be
random, depending on insertion preferences of
system
3) Integration into a Defined Landing
Site -Combine DNA of interest with integration
sequence (e.g. from bacteriophage) -Use host
whose genome has already been modified to contain
landing site -Supply DNA Integrase
4) Homologous Recombination/ Gene
Targeting -Homologous regions of DNA can be
targeted for recombination -Allows insertion of
DNA construct in defined site -Used to knock
out or modify gene of interest -Low frequency
event need to be able to screen large numbers of
events (e.g. in ES cells in culture)
14
Curent Methods of Choice
C. Elegans -inject DNA into syncytial
gonad -get multicopy extrachromosomal arrays of
DNA
Drosophila -inject DNA into posterior pole of
embryo (germ plasm) -use transposon backbone
transposase OR -phage integration site
integrase
Zebrafish -REMI -Transposase systems working
and gaining popularity
Mouse -inject DNA into oocyte nucleus for random
integration -transfect ES cells and screen for
homologous recombination when need targeted
insertion
15
Some Reasons for Wanting to Get DNA Into the
Genome
1) Identify New Genes 2) Drive Expression of a
gene of interest 3) Create a mutation in a gene
of interest
16
Transposons as Mutagens
Advantage mutated gene is tagged by transposon
for easy identification Disadvantage
transposons do not insert randomly -some genes
hit easily, others not
17
Modified Screening Vectors The Enhancer Trap
18
Modified Screening Vectors The Gene Trap
19
Modified Screening Vectors The Protein Trap
After Morin and Chia, PNAS 2001 Cooley and
Spradling Labs
20
Modified Screening Vectors The Secretory Trap
-splice acceptor -TM domain (selects for genes
with signal sequence) -ß-geo ß-gal-neo fusion
protein -IRES internal ribosome entry site -PLAP
placental alkaline ppase (labels neuronal axons)
21
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22
Reasons for Wanting to Get DNA Into the Genome
1) Identify New Genes 2) Drive Expression of a
gene of interest 3) Create a mutation in a gene
of interest
23
Reasons to drive expression of a gene of
interest -Observe an over-expression or dominant
negative phenotype -Rescue a mutant phenotype to
prove your gene is responsible -Conduct a
structure-function analysis of your
protein -Perform gene therapy
Ways to drive expression of your gene Desired
Expression Promoter Used Everywhere Viral
promoter or housekeeping (tubulin) At a
specific time Inducible (heat shock, hormone
responsive) In a specific place Tissue
specific Various places/times Exogenous
Binary System (e.g. Gal4/UAS or TET)
24
  • Tools for spatial and temporal control of gene
    expression
  • 1) Cell-type specific promoters to control
    expression at particular times and places
  • 2) Exogenous transcription factors that can also
    be conditional
  • Gal4 and Switch-Gal4 (activator)
  • Gal80 and TS Gal80 (repressor)
  • TET ON and OFF

25
Tissue-specific Promoter
100s of lines with different Gal4 patterns now
available
26
Modified Screening Vectors The Enhancer Trap
27
Gal4/UAS With Temporal Control
28
Gal4/UAS/TET With Temporal Control
29
Locking ON Gene Expression
Promoter 1
Gal4
UAS
Gal4
UAS
YFG
Once Gal4 has been produced from Promoter 1,
autoregulation through UAS-Gal4 will insure that
it stays on even if Promoter 1 doesnt. e.g.
Lineage tracing cells expressing Promoter 1
30
  • Tools for spatial and temporal control of gene
    expression
  • 1) Cell-type specific promoters to control
    expression at particular times and places
  • 2) Exogenous transcription factors that can also
    be conditional
  • Gal4 and Switch-Gal4 (activator)
  • Gal80 and TS Gal80 (repressor)
  • TET ON and OFF
  • 3) Site-specific recombinases that can also be
    conditional
  • FLP/FRT
  • CRE/LOX and CRE-ER (inducible CRE)

31
FLiPing Out
FRT sites (or loxP sites)
TXN Stop
(or CRE)
Struhl and Basler, 1993
Gene of interest only expressed after
recombinases excises transcription
STOP Recombinase can also be controlled spatially
(promoter-CRE construct) and temporally (hormone
inducible CRE recombinase)
32
Combinatorial Approaches Boolean Logic of Gene
Expression
Promoter 1
STOP
FRT
Switch-Gal4
Promoter 2
FLPase
UAS
YFG
YFG will be expressed wherever Promoter 2 is
active AND Promoter 1 is active AND when hormone
(RU486) is supplied
Similar combinations possible with CRE, inducible
CRE, TET ON and OFF, etc.
33
Some Reasons for Wanting to Get DNA Into the
Genome
1) Identify New Genes 2) Drive Expression of a
gene of interest 3) Create a mutation in a gene
of interest
34
More Fun With Recombinases Tissue Specific Gene
Inactivation
-Create allele of gene flanked by loxP sites
(floxed) (need one large or two smaller
targeting constructs) -Verify that floxed allele
still has wt activity -Generate the following
genotype
Gene Xfloxed
Cell-type specific Cre expression
Gene X?
-Region of gene b/w loxP sites is excised only
where Cre is expressed
Floxed allele
Deletion allele
35
Mitotic Recombination
Genetic mosaics can be created by mitotic
recombination induced by X-rays or a
site-specific recombinase
FLP/FRT in Drosophila Golic and Lindquist,
1989 Essential reagents Xu and Rubin, Chou and
Perrimon
Homozygous Mutant Daughter Cell
Heterozygous Mother Cell
J. Treisman
36
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39
Thummel, 1988
-Heat shock or constituitive expression -Promoter
analysis -Enhancer trapping -Insertion of FRT
sites into genome (Golic) -Gal4/UAS system (Brand
and Perrimon) -EP lines for random
over-expression (Rorth) -Other transposon systems
(PiggyBac, Minos)
40
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