Why clone in eukaryotes?

1. 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)

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. -- For large cells -- Can only transform a few cells at a time Direct DNA transfer --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 selectAGAINST 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 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 recombination 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 11;101(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 • The utility of embryonic stem (ES) cells • Transgenic animals (mainly mice)

46. 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

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 <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

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