Why clone in eukaryotes?

1
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)

2
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
• PROTOTYPICAL EUKARYOTE

3
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)

4
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

5
A yeast episomal plasmid
Shuttle vector has sequences allowing
replication in E.coli
6
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

7
YAC yeast artificial chromosome

• Replicates as chromosome has centromere and
  telomeres
• Useful for cloning very large pieces of DNA

8
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

9
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????
10

• 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.

11
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
12

• Yeast integrative plasmids
• Propagate and engineer using E. coli as a host
• No yeast origin of replication (MUST integrate)
• Genome engineering through homologous
  recombination

13

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

14
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
  function

15
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

16

• 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

17

• 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

18
Cationic lipids create artificial membranes that
bind to DNA. The lipids then bind to cell
membranes and fuse, delivering the DNA
19
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
20
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,
  adeno-associated virus have all been approved for
  clinical trials

21
Transient transformation (transfection)

• DNA maintained in nucleus for short time
• Extra-chromosomal, no replicon
• No selection is required

22
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

23
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

24
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
25
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

26
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

27
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)
28
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)
29

• Eukaryotic cell transformation
• Getting DNA in method depends on the type of
  cells
• Transient transformation no selection
• Stable transformation selection is required
  (also, counter-selection can be useful)

30
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

31
Introducing subtle mutations with minimal
footprints

• 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)

32
neo
Tag and exchange strategy
Tk
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
33
Considerations in homologous recombination
strategies

• 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

34
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

35
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
36
Cre expression induced by transient transfection
Diptheria toxin Prevents non-homologous recombina
tion
Selection and counter-selection markers flanked
by loxP sites
37
Recombinase activation of gene expression (RAGE)
loxP sites
Can be under conditional control
38
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)

39
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

40
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.
41

• 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
  siRNA?

42
Lentiviral vectors for expression of siRNA
43
Mouse embryo fibroblasts, infected with
lentiviruses (LV)
Cre recombinase
control
test
p65 tx factor
Targets of p65
controls
Western blots for specific proteins
44

• 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

45
Genetic manipulation of animals

1. The utility of embryonic stem (ES) cells
2. Transgenic animals (mainly mice)

46
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
47
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
  marker)

48
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)

49
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
50
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

x
51
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
  stem cells, and how do these signals function?
• Stem cell development could provide models for
  human tissue development

52

• 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,
  etc.)
• (SCID Severe combined immunodeficiency)

53

• Adult stem cells are multipotent (and possibly
  pluripotent?)
• 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

54

• 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

55
Retracted, 2005
56
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

57
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
58
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)
59
(No Transcript)
60
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?
61
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?
62
Never say die current efforts to create SCNT
clones
63
Other efforts to create ES cell lines
mice
64
Other efforts to create ES cell lines
mice
mice
65
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?)

66
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
67
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

68
Microinjection
Early embryo
Gentle suction
DNA
Pronucleus?
69
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

70
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

71
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

72
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)
73
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

74
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

75
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???

76
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)
77
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

78
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

79
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