Download Why clone in eukaryotes?

Cloning in S. cerevisiae
(cloning in eukaryotes, part 1)
Why clone in eukaryotes?
• Eukaryotic genes may not be expressed properly in
bacterial host
– different mechanisms for gene expression
– modifications (glycosylation)
• very large pieces of DNA can be cloned (yACs)
Why Saccharomyces cerevisiae?
1) easy to grow and manipulate (like E.coli)
2) biochemistry and cell biology similar between yeast
and “higher” eukaryotes
-- many gene homologs between yeast and humans, eg. Cell
cycle (cancer) genes
3) excellent genetic tools are available in yeast
“PROTOTYPICAL” EUKARYOTE
Yeast transformation
• Electroporation, or chemical competence (Lithium
chloride/PEG treatment)
• Isolate transformants using nutritional markers:
– His3, Leu2, Trp1--amino acid biosynthetic genes
– Ura3--nucleotide biosynthetic gene
(these require auxotrophic yeast strains)
– Aminoglycoside (ribosome inactivating) antibiotic
resistance (kanamycin)
YEp: high copy number plasmid
• Yeast Episomal plasmid
• Contains naturally occuring “2 micron circle”
origin of replication
• High copy number: 50-100/cell
• Shuttle vector -- replicon for E. coli
A yeast episomal plasmid
Shuttle vector: has
sequences allowing
replication in E.coli
YCp: low copy number plasmid
• Yeast Centromeric plasmid
• Contains yeast ars (autonomously replicating
sequence) for replication
• Contains yeast centromere for proper
segregation to daughter cells
• Low copy number, ~1 per cell (good for cloning
genes that are toxic or otherwise affect cell
physiology)
• Stable, shows Mendelian segregation
YAC: yeast artificial chromosome
• Replicates as
chromosome:
has centromere
and telomeres
• Useful for
cloning very
large pieces of
DNA
Yeast integrative plasmid:
homologous recombination
• No yeast replicon, can transform but cannot
replicate
• Requires integration into chromosome for
propagation, but very stable
• Useful for manipulating (eg. deleting) genes
on the chromosome
The first demonstration of a yeast integrative
plasmid: leu2 complementation
Wild type yeast: grows on minimal medium lacking leucine
because it has the leucine biosynthetic genes
Leu2 yeast: a mutation in the leu2 gene, it knocks out leucine
biosynthesis, therefore no growth without leucine
pYeLeu10: a plasmid (with no yeast replicon) that contains
the yeast Leu2 gene--can it complement the Leu2 mutant
yeast????
The experiment:
•Transform Leu2 mutant cells, using pYeLeu10
(which contains an intact Leu2 gene)
•select for growth in the absence of leucine (leu
dropout plates)
•What will grow? Only those cells that can replicate
the Leu2 gene coming from the plasmid
Results: some transformants survive….
Three ways for the leu2 gene to be maintained
(all via integration)
Mutant Leu2
1) Double
crossover
(3 kinds)
2) Single
crossover
(integration)
3) Random
insertion
Yeast integrative plasmids
1) Propagate and engineer using E. coli as a host
2) No yeast origin of replication (MUST integrate)
3) Genome engineering through homologous
recombination
Gene transfer to animal cells
A. DNA transfer methods
B. Non-replicative transformation (transient
transfection)
C. Stable transformation
Readings: #32
Gene transfer to animal cells--why?
• Animal cell culture useful for production of
recombinant animal proteins: accurate posttranslational modifications
• Excellent tool for studying the cell biology of
complex eukaryotes
– Isolated cells, simplifies analysis
– Human cell lines: a way of studying human cell
biology without ethical problems
• Establish conditions for gene therapy--treatment of
genetic disorders by restoration of gene function
Strategies for gene transfer
• Transfection
– Cells take up DNA from medium
• Direct transfer
– Microinjection into nucleus
– “gene gun”: particles coated with DNA
bombarding cells
• Transduction
– Viral mechanism for transfer of DNA to cells
Transfection by DNA/Calcium phosphate
coprecipitate
– Mammalian cells will take up DNA with this
method--endocytosis of the precipitate?
– Only suitable for cell monolayers, not cell
suspensions
– Up to 20% of cells take up DNA
Liposome-mediated transformation
(lipofection)
– Liposomes--artificial phospholipid vesicles
– Cationic/neutral lipid mixtures spontaneously
form stable complexes with DNA
– Liposomes interact with negatively charged cell
membranes and the DNA is taken up by
endocytosis
– Low toxicity, works for most cell types, works
with cells in suspension
– Up to 90% of cells can be transfected
Cationic lipids create
artificial membranes that
bind to DNA.
The lipids then bind to cell
membranes and fuse,
delivering the DNA
Direct DNA
transfer
--Works well on
tissues, plant cells
These methods
are used when
other (easier)
methods fail
-- For large cells
-- Can only transform a
few cells at a time
Viral transduction
• Exploiting viral lifestyle (attachment to cells and
introduction of genomic DNA) to introduce
recombinant DNA
• Transfer genes to cultured cells or to living
animals
• Potentially useful in gene therapy
– Retrovirus, adenovirus, herpesvirus, adenoassociated virus have all been approved for
clinical trials
Transient transformation (transfection)
• DNA maintained in nucleus for short time
• Extra-chromosomal, no replicon
• No selection is required
How is transient transformation useful?
• Testing platform prior to time-consuming and
difficult cell-line construction
• Experiments: e.g. investigating gene regulatory
regions
– Clone regulatory elements upstream of a
reporter gene on plasmid
– Chloramphenicol acetyl transferase (CAT) gene
activity varying depending on the levels of
transcription directed by regulatory elements
Stable transformation
• A small fraction of the DNA may be integrated into
the genome--these events lead to stable
transformation
• Homologous recombination can be exploited for
genome engineering
• Results in formation of a “cell line” that carries
and expresses the transgene indefinitely
• Selectable markers greatly assist in isolating
these rare events
Mysteries of stable transfection/
transformation
Mechanism of transport of DNA is not known: “Some
DNA” is transported to the nucleus
Non-homologous intermolecular ligation events may occur
Large concatameric rDNA structure may eventually
integrate, usually by non-homologous recombination
Best case scenario: 1 in 1000 transfected cells will carry
the transfected gene in a stable fashion
Selectable markers for transformation:
“Dominant” selectable markers
• Confer resistance to some toxin, eg. the neo
marker (neomycin resistance) confers survival in
presence of aminoglycoside antibiotics
–
–
–
–
Kanamycin
Bleomycin
“G418” (dominant selectable marker)
These antibiotics affect both bacterial and eukaryotic
protein synthesis
• These selectable markers do not require a
specific genotype in the transfected cell-line
Selectable markers for transformation:
endogenous markers
• Confer a property that is normally present in
cells, eg. thymidine kinase (TK) (required for
salvage pathway of nucleotide biosynthesis)
• These markers may only be used with cell
lines that already contain mutations in the
marker genes
Thymidine Kinase gene: a selectable marker
Grow thymidine kinase knockout cells in HAT medium
(hypoxanthine,aminopterin, and thymidine)
Aminopterin blocks de novo synthesis of TMP and A/GMP
(restore A/GMP synthesis with hypoxanthine),
thymidine for salvage pathway (requires thymidine kinase)
Counter-selectable markers
You can select AGAINST thymidine kinase, by treating
Tk+ cells with TOXIC nucleotide analogues that are
only incorporated into DNA in by thymidine kinase
examples:
5-bromo-deoxyuridine
Ganciclovir
Cells with TK die in the presence of these compounds,
Cells that lose the Tk gene survive
(the diptheria toxin gene, dipA, is also used in counterselection)
Eukaryotic cell transformation:
1) Getting DNA in: method depends on the type
of cells
2) Transient transformation: no selection
3) Stable transformation: selection is required
(also, counter-selection can be useful)
Applications of gene targeting
• Homozygous, null mutants (“knock-out”
mice): what is the effect on the organism?
• Correction of mutated genes: gene therapy
(confirming genetic origin of a disease)
• Exchange of one gene for another (gene
“knock-in”)
– Example: exchange parts of mouse
immune system with human immune
system
Introducing “subtle” mutations with
minimal footprints
Two steps:
1) Target gene by homologous
recombination
2) Remove or replace selection marker
gene by counter selection (e.g. thymidine
kinase gene is lethal in the presence of
toxic thymidine analogs like ganciclovir)
neo
Tk
“Tag and exchange”
strategy
First transformation,
select for neo
Tk
neo
Counter-selection:
select against Tk gene
by adding ganciclovir
(lethal nucleotide, only
incorporated into the cell
in the presence of Tk)
Tk
neo
Very clean strategy, no
markers are introduced
Considerations in homologous
recombination strategies
•
Random insertion of DNA often occurs--how
to get around this problem?
1) Add a negative selection gene to the DNA
outside of the region of homology (ensure
that the cells containing this gene via nonspecific integration will die)
2) Screen transformants by PCR for correct
position of recombinant DNA insertion
Site-specific recombination
• Specialized machinery governs process
• Recombination occurs at short, specific
recognition sites
Homologous recombination
• Ubiquitous process
• Requires long regions of homology between
recombining DNAs
Cre-Lox (site-specific) recombination
• Cre is a protein that catalyzes the recombination
process (recombinase)
• LoxP sites: DNA sequences recognized by the Cre
recombinase
Direct
repeats:
Deletion of
intervening
sequences
Inverted repeats:
inversion
Selection and
counter-selection
markers flanked
by loxP sites
Diptheria toxin:
Prevents nonhomologous
recombination
Cre expression
induced by
transient
transfection
Recombinase activation of gene expression
(RAGE)
loxP sites
Can be under
conditional
control
Cre-mediated conditional
deletions in mice
• Surround gene of interest with lox sites (gene is
then “floxed”)
• Place Cre gene under inducible control
• Gene of interest can be deleted whenever
necessary (allows study of deletions that are
lethal in embryo stage)
Strategies for gene inhibition
• Antisense RNA transgenes: synthesize
complement to mRNA, prevent expression of
that gene
• RNA interference (RNAi): short doublestranded RNAs (siRNAs) silence gene of
interest--can be made by transgenes or
injected, or (in the case of C. elegans) by
soaking in a solution of dsRNA
• Intracellular antibody inhibition: transgene
expresses antibody protein, antibody binds
protein of interest, inhibits expression
Paper:
CRE recombinase-inducible RNA interference
mediated by lentiviral vectors.
Tiscornia G, Tergaonkar V, Galimi F, Verma IM.
Proc Natl Acad Sci U S A. 2004 May
11;101(19):7347-51. Epub 2004 Apr 30.
Background of this paper
1) Alternatives to simple gene knockouts are desirable,
regulated gene knockout is valuable
2) Gene activity can be turned off by the activity of small
interfering RNA (siRNA), which inactivates mRNA
through complementarity and an RNA-induced silencing
complex (RISC, a nuclease)
3) siRNA can be delivered by lentiviral (modified retrovirus)
vectors
This paper attempts the controlled expression of siRNA by
separating the siRNA from its promoter with transcription
terminators flanked by loxP sites: can CRE recombinase
expression induce siRNA?
Lentiviral vectors for expression of siRNA
Mouse embryo fibroblasts, infected with lentiviruses (LV)
Cre recombinase
control
test
p65 tx factor
Targets of p65
controls
Western blots for specific proteins
Results:
1) An inducible gene knockout without recombination
(requires two separate lentiviral vectors, simultaneous
infection with both vectors)
2) If CRE is expressed in “tissue-specific” backgrounds,
can study gene knockout in specific tissues (rather than
systemic knockouts)
3) Allows the study of genes that are “embryonic-lethal”
when knocked out normally
Genetic manipulation of
animals
1) The utility of embryonic stem (ES)
cells
2) Transgenic animals (mainly mice)
Methods for
generating transgenic
animals
Terminology
Transgenic: all cells in the
animal’s body contain the
transgene, heritable (germ
line)
Chimeric: only some cells
contain the transgene, not
heritable if the germ line is
not transgenic
Gene targeting with ES cells
• Introduction of specific mutations to ES cell
genome
• Transform with linearized, non-replicating
vector containing DNA homologous to target
DNA region, look for stable transfection
• Use positive selection to obtain homologous
recombinants, e.g. the neo marker (neomycin
resistance, confers survival of aminoglycoside
antibiotics like “G418” (dominant selectable
Stem cells--what are they?
• Unspecialized, undifferentiated cells
• “Renewable” through cell divisions, capable of
dividing many times
• Can be induced to differentiate into specialized cell
types, e.g. cardiac, neural, skin, etc.
Two types:
– Embryonic stem (ES) cells: from embryos,
pluripotent (giving rise to any cell type), also
totipotent? (able to develop into a new individual
organism?)
Totipotent: capable of developing into a complete
organism or differentiating into any of its cells or
tissues <totipotent blastomeres>
Pluripotent: not fixed as to developmental
potentialities : having developmental plasticity
<pluripotent stem cell>
Multipotent: not a real word (Merriam Webster), but it
refers to adult stem cells that can replenish cells of a
specific type, example: hematopoeitic stem cells
Sources of stem cells?
• ES cells: from inner cell mass of early
embryo
– human ES cells first cultured in 1998, using
donated embryos (with consent) created for
fertility purposes
– ES cells from cloned somatic cells (2004)
x
• AS cells: from adult tissues
Some politics come into play here
Usefulness of stem cells
• Medical:
– ES cells are pluripotent, and could be used to
produce new tissues for “regenerative” medicine
– Cloned ES cells could be used to generate cells
and tissues that would not be rejected by the
recipient
– ES-derived cell types could be used in toxicity
testing
• Scientific
– How do stem cells remain unspecialized in
culture?
– What are the signals that cause specialization in
How do you know if you have ES cells?
1) Growth capacity: ES cells are capable of lots of cell
divisions in culture without differentiation
2) Cell-type “markers” tell you what kind of a cell you have:
Oct-4 protein expression is high in ES cells but not in
differentiated cells
3) Chromosomes should be normal: Check the karyotype
(many immortalized cell lines are cancer-derived, and
often have abnormal karyotypes)
4) The cells must be differentiatable
A) Allow natural differentiation
B) Induce differentiation
C) Check for teratoma formation in SCID mice
(Teratoma: “benign” tumor containing all cell types in
a jumble, often containing hair, teeth, etc.)
(SCID: Severe combined immunodeficiency)
Adult stem cells are multipotent (and
possibly pluripotent?)
1) hematopoeitic: blood cells
2) bone marrow stromal cells: bone, cartilage,
connective tissue, fat cells
3) neural: brain and nerve cells
4) epithelial: cells lining the digestive tract
5) skin: epidermis, follicles
6) Germ-line cells: sperm, eggs
But some of these stem cell types can do more:
brain stem cells can differentiate into blood and
skeletal muscle cells
ES versus AS cells? Some important differences
•ES cells are pluripotent
•AS cells are generally limited to the tissue type that they came
from
•ES cells divide a lot in culture (easy to manipulate and
propagate)
•AS cells are very rare, generally difficult to isolate, and at this
time cannot be cultured
Retracted, 2005
The idea:
• Adult cell provides nucleus
• Enucleated egg (donated) provides
cytoplasm
– (Somatic Cell Nuclear Transfer--SCNT)
• Newly diploid egg begins to divide, forming
an embryo
• The embryo develops to blastocyst stage
• ES cells are taken from the inner cell mass,
destroying the clone embryo
RETRACTED Conclusions:
Human ES cells can be derived by SCNT (cloning)
cells can divide for a long time
cells can differentiate
cells display ES cell markers
cells can form teratomas
Potential positive implications of this
research:
-- Another source of human ES cell lines (not a traditionally
derived embryo)
-- Suggests a way to generate tissues or cell types that would
be host-derived and so would not be rejected by the patient
(but you still require oocytes)
-- Suggests a novel path for gene therapy: the somatic
genome can be manipulated in culture (using the same
techniques discussed for mouse ES cells) to correct genetic
aberrations, and the altered cells can be used in patientspecific treatments (seems expensive and time-consuming at
this time)
Other things to consider:
-- Would cloned ES cells be totipotent (giving rise to a whole
person)? Would anyone attempt to clone a human? Why?
Would a cloned person develop properly, live a normal life?
-- How would long term use of ES cell-derived medical
therapy affect lifespan, quality of life, survival/evolution of the
species?
What about the eggs required for transfer?
Human eggs have a limited availability
Egg donation is not trivial--a potentially risky medical
procedure
Should egg donors be paid?
Can human eggs be produced by animal chimeras?
Never say die: current efforts to create SCNT clones
Other efforts to create ES cell lines:
mice
Other efforts to create ES cell lines:
mice
mice
Alternatives to embryos as source for “ES-like” cells?
•Mouse testis: source of spermatogonial stem cells (SSCs)
•SSCs can acquire embryonic stem cell properties
•Name: Multipotent adult germline stem cells (maGSCs)
•Properties:
•differentiation into 3 embryonic germ layers
•generate teratomas
•when injected into blastocyst, they contribute to
development of organs and germline
•No SCNT required
•Potential source of therapeutic stem cells
•(oogonial stem cells too?)
Methods for
generating transgenic
animals
Terminology
Transgenic: all cells in the
animal’s body contain the
transgene, heritable (germ
line)
Chimeric: only some cells
contain the transgene, not
heritable if the germ line is
not transgenic
Producing transgenic mice
• Pronuclear microinjection--an early technique
– Immediately following fertilization, male (sperm)
pronucleus is large and is the target for microinjection
– Arrays of the recombinant DNA molecule can form prior
to integration
– DNA may integrate immediately (transgenic) or may
remain extrachromosomal for one or more cell divisions
(chimeric)
– Site of DNA integration apparently random
– Chromosomal rearrangements and deletions
– POOR CONTROL
Microinjection
Early embryo
Gentle suction
DNA
Pronucleus?
Intracytoplasmic sperm
injection
• Plasmid DNA binds to sperm heads in
vitro
• Inject DNA-coated sperm heads into
egg
• integration of the carried plasmid DNA
along with fertilization of the egg by the
sperm
Somatic cell nuclear transfer
• Donor diploid nucleus isolated from various cell
types, including adult somatic cells
• Nucleus injected into enucleated egg cells
• Clones of animals (frogs in the 1950s, mammals
in the 1990s)
• Difficult procedure: the donated nucleus needs to
be synchronized at the level of cell cycle with the
acceptor egg cell
• Earlier stage (less differentiated) donated nuclei
work best
• High rates of failure with this protocol
Recombinant retrovirus
transduction
***Retroviruses are RNA viruses that replicate
via a double-stranded DNA intermediate,
which is stably integrated into the host
genome at random positions
• Infect preimplantation embryos or embryonic
stem (ES) cells
Retroviruses as tools for engineering
--RNA viruses
--Double-stranded DNA intermediate integrates into
genome (semi-randomly)
--Single integrated copy in genome, stable
--Some infect only dividing cells
--Maximum transgene capacity is about 8 kbp (viral
genes are replaced, and helper virus is required)
Producing transgenic mice
• Embryonic stem (ES) cell transfection
***ES cells are derived from mouse blastocyst
(early embryo) and can develop into all cell
types, including germ line (totipotent)
• ES cells can be propagated in culture and
transformed by all methods described for
animal cells using standard markers
• ES cells then can be moved to blastocyst for
development
Are the mice truly transgenic?
• Recombinant ES cells (from agouti mice,
dominant coat color) introduced to host
(recessive black coat color) blastocyst,
progeny screened for chimerics (both
black and agouti)
• Chimeric male progeny are mated to
black-coat females, any agouti offspring
confirm the presence of the transgene in
the germline
Transgenic mice: controlling
gene expression in the
organism
• Regulatory region of mouse
metallothionein-1 gene (MMT-1) is induced
in response to heavy metals (Cd, Zn, etc.)
• Induce other genes by fusing them to
MMT-1 regulatory region???
MMT-1 promoter fused to rat growth hormone gene
Without
fusion
With
fusion
But:
-- a lot of variability in expression from mouse to mouse: position
effects, gene expression is highly dependent on chromosomal
context of the integrated transgene
-- progeny of transgenic mice had unpredictable expression of
MMT-1/rat growth hormone fusion (not a stable phenotype)
Position effects in transgene
insertion
• Local regulatory region of DNA is very
important
• Chromatin structure can be repressive
(silencing by heterochromatin)
• Defeat position effects by
– Include gene plus DNA upstream and
downstream
– Include specific regulatory sequences
(locus control region (LCR), boundary
elements to prevent silencing of gene
expression
– Include introns
YAC transgenic mice
• Sometimes it is necessary to transfer
very large pieces of DNA to the mouse,
e.g. the human HPRT gene locus
(which almost 700 kilobases long)
• YACs (yeast artificial chromosomes)
work well for this, ES cells may be
transformed by lipofection
Transgenics in other mammals and
birds
• Traditional techniques for mice have had mixed
success
– Efficiency of pronuclear transfer is generally very low
– Retrovirus-induced transgenic animals have been isolated,
but this is also inefficient
– Very very difficult to derive reliable ES cell lines from any
domestic species besides mice, chickens (although human
ES cell lines are available)
– Thus, less sophisticated techniques are all that is possible