Why do we care about homologous recombination?

1. Why do we care about homologous recombination? • Universal biological mechanism • Bacteria can pick up new genes • Biotechnology • Gene knockouts in mice via homologous recombination

2. DNA of interest in mouse chromosome This is the gene targeted for replacement by an engineered construct. Note flanking upstream and downstream DNA sequences. The arrows pointing away from the targeted gene represent the continuous chromosomal DNA

3. 1. Prepare construct DNA in lab with selectable marker Engineered construct used to replace the gene. Upstream and downstream flanking DNA sequences are identical to those which flank the targeted gene.

4. 2. Add construct to embryonic stem cells (ES) in culture Amazingly, the DNA construct finds its way into ES cell nucleus and aligns itself with targeted gene.

5. 3. homologous recombination by cell The chromosome now contains engineered construct in place of the original allele. The original allele has been recombined into the construct and is lost over time.

6. 4. Add ES cells to embryo  implant in surrogate mother 5. Cross breed to create homozygous knockout

7. Back to bacteria….. • Hfr strains led to mapping of the E. coli chromosome • Interrupted mating technique to map genes on E. coli

8. Lederberg’s experiment explained

9. Fig. 15.7 Hfr H(aziRtonRlac+gal+strS) X F- (aziStonSlac-gal-strR)

10. Circular chromosome 4.6 million bp (4.6 Mb)

11. 2. Transformation • Naked DNA enters bacterial cell. Brings new genes (can change bacteria phenotype) • Bacterium with new DNA is a transformant

12. Transformation (rare event) • Natural flash animation • Engineered • CaCl2 treat bacteria  competent cells • cell membrane permeable to naked DNA

13. Plasmids can be cloning vectors Ch 8 pg 175 • pUC19 ampr gene ori restriction sites (multiple cloning site)

14. Plasmid requirements in biotech • Ori for DNA replication • Selectable marker ex. ampr • Only cells that take up the plasmid are resistant to amp • Restriction enzyme sites • High copy number in E. coli (100/cell)

15. Shimomura Ampr Ori araC GFP

16. Viruses can bring new genes into a cell

17. Transduction –phage mediated transfer of genes into bacteria • Bacteriophage – virus that infects bacteria • Lederberg and Zinder 1952

18. phage • DNA or RNA surrounded by protein coat • genes encode for viral activity, viral parts

19. Viral infection lytic cycle 1. Virus adsorbs to cell and injects DNA

21. 2. normal bacterial activity is shut down and bacterium becomes a “phage factory”

23. 3. host DNA broken into pieces, new viruses released to infect new cells

24. chromosomal DNA is chopped as viruses destroy cell

25. Generalized transduction • A piece of chromosomal DNA gets packaged into a virus = faulty head stuffing • This transducingphage infects a new cell and transfers genes from the first bacterium • Homologous recombination occurs • Flash animation generalized transduction

27. Bacteriophage phenotypes • virulent phage - always lytic, cannot become a prophage • temperate phage - lysogenic

28. Temperate phage and lysogenic pathway • Phage DNA integrated into specific location in chromosome • Prophage is lysogenic • Phage gene represses lytic cycle • Flash animation specialized transduction

29. Growing phage • Grow bacterial lawn on agar dish • Add phage  infects bacteria • Obtain plaques (where cells have lysed) • Obtain phage lysate (contains phage)

30. plaques

31. Gene therapy with virus (Ch 10) • Objective : insert normal gene into human DNA • Candidates: people with single gene disorders • Use virus as vector Adenovirus. Child Health and Human Development Bioinformation video

32. Gene Therapy ADA 1990 • Gene for adenosine deaminase • ADA normally eliminates deoxyadenosine (from degraded DNA) (recessive disease) • dA toxic to lymphocytes •  Severe immune deficiency

33. Ashanti Disilva was 4 and dying • remove viral replication genes • insert normal ADA gene into virus • remove T cells from patient • infect cells with engineered virus • infuse into patient

34. Problems with gene therapy • Inflammatory response to virus  death • Gene disrupts cell cycle gene cancer • Other methods • Liposomes • Stem cells