1. Gene Transfer BY NIKAM C.D. ASSISTANT PROFESSOR,
DEPARTMENT OF MICROBIOLOGY,
S.M. JOSHI COLLEGE, HADAPSAR, PUNE

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

3. 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

4. 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.

5. 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.

6. 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.

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

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

9. Lederberg’s experiment explained

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

11. Circular chromosome
4.6 million bp (4.6 Mb)

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

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

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

15. 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)

16. Shimomura
Ampr
Ori
araC
GFP

17. Viruses can bring new genes into a cell

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

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

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

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

24. 3. host DNA broken into pieces, new viruses released to infect new cells
chromosomal DNA is chopped as viruses destroy cell

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

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

29. 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

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

31. plaques

32. 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

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

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

35. Than you