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Cloning in S. cerevisiae (cloning in eukaryotes, part 1) Why


Cloning in S. cerevisiae (cloning in eukaryotes, part 1) Why clone in eukaryotes? Eukaryotic genes may not be expressed properly in bacterial host – PowerPoint PPT presentation

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Title: Cloning in S. cerevisiae (cloning in eukaryotes, part 1) Why

Why clone in eukaryotes?
Cloning in S. cerevisiae (cloning in eukaryotes,
part 1)
  • 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?
  • easy to grow and manipulate (like E.coli)
  • biochemistry and cell biology similar between
    yeast and higher eukaryotes
  • -- many gene homologs between yeast and humans,
    eg. Cell cycle (cancer) genes
  • excellent genetic tools are available in yeast

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
  • Stable, shows Mendelian segregation

YAC yeast artificial chromosome
  • Replicates as chromosome has centromere and
  • Useful for cloning very large pieces of DNA

Yeast integrative plasmid homologous
  • No yeast replicon, can transform but cannot
  • 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
2) Single crossover (integration)
(3 kinds)
3) Random insertion
  • Yeast integrative plasmids
  • Propagate and engineer using E. coli as a host
  • No yeast origin of replication (MUST integrate)
  • Genome engineering through homologous

  • Gene transfer to animal cells
  • A. DNA transfer methods
  • B. Non-replicative transformation (transient
  • C. Stable transformation
  • Readings 32

Gene transfer to animal cells--why?
  • Animal cell culture useful for production of
    recombinant animal proteins accurate
    post-translational 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

Strategies for gene transfer
  • Transfection
  • Cells take up DNA from medium
  • Direct transfer
  • Microinjection into nucleus
  • gene gun particles coated with DNA bombarding
  • Transduction
  • Viral mechanism for transfer of DNA to cells

  • Transfection by DNA/Calcium phosphate
  • Mammalian cells will take up DNA with this
    method--endocytosis of the precipitate?
  • Only suitable for cell monolayers, not cell
  • 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
-- For large cells -- Can only transform a few
cells at a time
--Works well on tissues, plant cells These
methods are used when other (easier) methods fail
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
  • Potentially useful in gene therapy
  • Retrovirus, adenovirus, herpesvirus,
    adeno-associated 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
  • 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
  • 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
  • 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-deoxyurid
ine 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 counter-selection)
  • Eukaryotic cell transformation
  • Getting DNA in method depends on the type of
  • Transient transformation no selection
  • 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
  • Example exchange parts of mouse immune system
    with human immune system

Introducing subtle mutations with minimal
  • Two steps
  • Target gene by homologous recombination
  • 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)

Tag and exchange strategy
First transformation, select for neo
Counter-selection select against Tk gene by
adding ganciclovir (lethal nucleotide, only
incorporated into the cell in the presence of Tk)
Very clean strategy, no markers are introduced
Considerations in homologous recombination
  • Random insertion of DNA often occurs--how to get
    around this problem?
  • Add a negative selection gene to the DNA outside
    of the region of homology (ensure that the cells
    containing this gene via non-specific integration
    will die)
  • 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

Direct repeats Deletion of intervening sequences
Inverted repeats inversion
Cre expression induced by transient transfection
Diptheria toxin Prevents non-homologous recombina
Selection and counter-selection markers flanked
by loxP sites
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 double-stranded
    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 11101(19)7347-51. Epub
2004 Apr 30.
  • Background of this paper
  • Alternatives to simple gene knockouts are
    desirable, regulated gene knockout is valuable
  • 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)
  • 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

Lentiviral vectors for expression of siRNA
Mouse embryo fibroblasts, infected with
lentiviruses (LV)
Cre recombinase
p65 tx factor
Targets of p65
Western blots for specific proteins
  • Results
  • An inducible gene knockout without recombination
    (requires two separate lentiviral vectors,
    simultaneous infection with both vectors)
  • If CRE is expressed in tissue-specific
    backgrounds, can study gene knockout in specific
    tissues (rather than systemic knockouts)
  • Allows the study of genes that are
    embryonic-lethal when knocked out normally

Genetic manipulation of animals
  • The utility of embryonic stem (ES) cells
  • Transgenic animals (mainly mice)

Methods for generating transgenic animals
Terminology Transgenic all cells in the
animals 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
  • 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?)
  • Adult stem (AS) cells from adult tissues,
    multipotent (giving rise to specific cell types)

Totipotent capable of developing into a complete
organism or differentiating into any of its cells
or tissues lttotipotent blastomeresgt Pluripotent
not fixed as to developmental potentialities
having developmental plasticity ltpluripotent stem
cellgt 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)
  • 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
  • ES-derived cell types could be used in toxicity
  • Scientific
  • How do stem cells remain unspecialized in
  • What are the signals that cause specialization in
    stem cells, and how do these signals function?
  • Stem cell development could provide models for
    human tissue development

  • How do you know if you have ES cells?
  • Growth capacity ES cells are capable of lots of
    cell divisions in culture without differentiation
  • 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
  • Chromosomes should be normal Check the karyotype
    (many immortalized cell lines are cancer-derived,
    and often have abnormal karyotypes)
  • The cells must be differentiatable
  • Allow natural differentiation
  • Induce differentiation
  • Check for teratoma formation in SCID mice
  • (Teratoma benign tumor containing all cell
    types in a jumble, often containing hair, teeth,
  • (SCID Severe combined immunodeficiency)

  • Adult stem cells are multipotent (and possibly
  • hematopoeitic blood cells
  • bone marrow stromal cells bone, cartilage,
    connective tissue, fat cells
  • neural brain and nerve cells
  • epithelial cells lining the digestive tract
  • skin epidermis, follicles
  • 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
  • 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
patient-specific treatments (seems expensive and
time-consuming at this time)
(No Transcript)
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
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
Other efforts to create ES cell lines
Other efforts to create ES cell lines
Alternatives to embryos as source for ES-like
  • Mouse testis source of spermatogonial stem cells
  • SSCs can acquire embryonic stem cell properties
  • Name Multipotent adult germline stem cells
  • 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
animals 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
  • 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

Early embryo
Gentle suction
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
  • 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

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
  • 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
  • Thus, less sophisticated techniques are all that
    is possible