Why clone in eukaryotes? - Portland State University

Why clone in eukaryotes? - Portland State University

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, 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 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 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 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 Tk 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 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 non-specific 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 counterselection

markers flanked Diptheria toxin: Prevents nonhomologous recombination Cre expression induced by transient

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 RNAinduced 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 embryoniclethal 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

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 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)

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 Pluripotent: not fixed as to developmental potentialities : having developmental plasticity 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 x 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 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

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, 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 patient-specific 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 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 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 fusio n 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

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