Manipulating Genes and Cells

1. Manipulating Genes and Cells Chapter 10

2. In the early 1970s, it became possible, for the first time, to isolate a given piece of DNA out of the many millions of nucleotide pairs in a typical chromosome. • This in turns made it possible to generate new DNA molecules in the test tube and to introduce this genetic materials back into living organisms. • These developments, called “recombinant DNA,” “gene splicing,” or “genetic engineering” make it possible to create chromosomes with combination of genes that that could never have formed naturally–or combinations of genes that could conceivably occur in nature but might take thousands of years of chance events to come together.

3. Humans have been experimenting with DNA, albeit without realizing it for millennia. 10_01_experiment.DNA .jpg wild rose → modern garden rose gray wolf → modern dog

4. 10_02_cell_sorter.jpg fluorescence-activated cell sorter to obtain a uniform population of cells

5. in vitro, in vivo • in vitro: literally, “in glass” • in vivo: literally, “in the living” • experiments performed using cultured cells are sometimes said to be carried out in vitroto contrast them with experiments on intact organisms, which are said to be carried out in vivo *be often used in a very different sense by biochemists. • in vitro: reactions carried out in a test tube in the absence of cells • in vivo:reactions taking place inside a living cell p. 325

6. 10_03_Cells_origin.jpg oligodendrocytes fibroblasts myoblasts • primary culture • cell line: (immortalized) • embryonic stem (ES) cell lines: (undifferentiated) Tobacco cells

7. Restriction (endo)nucleases 10_04_Restrict.nuclease.jpg blunt end • sticky end • protruding end • cohesive end

8. Ethidium bromomide (EtBr) Sypro green, yellow,… 10_05_gel.electrophor.jpg • physical map • restriction map

9. 10_06_budding.yeast.S.jpg The complete nucleotide sequence of the budding yeast Saccharomyces cerevisiae was finished in 1996.

10. DNA sequencing: dideoxy method 10_07_1_enzym.dideoxy.jpg dideoxy method In vitro DNA polymerization chain-terminating by dideoxyribonucleoside triphosphate

11. 10_07_2_enzym.dideoxy.jpg denature acrylamide gel

12. Automated sequencing 10_08_DNA.sequencing .jpg genome sequencing → prediction ORFs (genes) →annotation → identification

13. Sequencing the human genome 10_09_Shotgun.sequenc.jpg shotgun sequencing

14. 10_10_Repetit.sequence.jpg

15. clone-by-clone 10_11_BAC.clones.jpg “fingerprints” 100 to 200 kbp cloned into BAC

16. 10_12_de_renaturation.jpg (hybridization) DNA/DNA; RNA/RNA; RNA/DNA Probe: 10- 1000 nt long

17. Human sickle-cell anemia, point mutation, recessive mutation 10_13_hybridization.jpg probe length hybridization temperature

18. 10_14_1_Southrn.blotting.jpg

19. 10_14_2_Southrn.blotting.jpg

20. Southern blotting- • DNA/DNA • Northern blotting- • DNA (RNA)/RNA • Western blotting- • protein/protein (Ab/Ag)

21. 10_15_DNA.microarrays .jpg (dot blot hybridization)

22. 10_16_In.situ.hybrdztn.jpg metaphase chromose 5 (Fluoresence) in situ hybridization (FISH)

23. 10_17_mRNA.in.situ.jpg in situ hybridization of cyclin expression cyclin: a protein triggers the cell to divide

24. DNA cloning • the act of making many identical copies of a DNA molecule • the separation of a particular stretch of DNA (gene) from the rest of a cells DNA • one of the most important feats of recombinant DNA technology

25. 10_18_ DNA.in.vitro.jpg

26. 10_19_DNA.uptake.jpg other bacterial cells that have died transformation

27. 10_20_Bacteria.plasmid.jpg • ds DNA • containing ori • carrying genes that render their host bacteria resistant to antibiotics (selectable porperty) • having cutting sites for restriction nucleases plasmid: carrier, vector

28. DNA cloning 10_21_DNA ligase.jpg

29. 10_22_cloned.DNA.frag.jpg

30. 10_23_genomic.library.jpg DNA library

31. 10_24_hybridization.jpg colony hybridization

32. 10_25_cDNA.jpg

33. genomic library V.S. cDNA library

34. 10_26_Genomic_cDNA.jpg

35. 10_27_1_PCR_amplify.jpg PCR (polymerase chain reaction) denaturation annealing extension

36. PCR: in vitro DNA replication 10_27_2_PCR_amplify.jpg 2n The key of PCR: thermal stable DNA polymerase

37. Applications of PCR • The method of choice for cloning specific DNA fragments • Detection of infections by pathogens at very early stages (for many infections, PCR is the most sensitive method of detection) • Having great potential in forensic medicine

38. DNA/gene cloning by PCR 10_28_PCR_clones.jpg

39. Detection of infections by pathogens 10_29_PCR_viral.jpg

40. Application of PCR in forensic medicine 10_30_1_PCR_forensic.jpg Hypervariable microsatellite sequences VNTR: Variable Number of Tandem Repeats →as a distinctive DNA fingerprint for each individual

41. 10_30_2_PCR_forensic.jpg When examining the variability at 5-10 different VNTR loci, the odds that two individuals would share the same fingerprint by chance are ~1/10,000,000,000

42. 10_31_SerialDNA.clone.jpg

43. 10_32_expressionvector.jpg

44. 10_33_gene_protein.jpg

45. 10_34_Reporter.genes.jpg commonly used reporter proteins in eucaryotic cells: β-galactosidase, GFP (green fluorescent protein)

46. 10_35_GFP.jpg tissue specific promoter + GFP

47. 10_36_mutagenesis.jpg site-directed mutagenesis

48. 10_37_engineered.org.jpg • transgenic organisms: organisms into which a new gene has been introduced, or those whose genomes have been altered in other ways using recombinant DNA techniques. • in vitro mutated DNA fragment introduced into organisms (or cells) • mutated geneinserted into the genome of an organism (or cell) via homologous recombination

49. RNA interference (RNAi) • introducing into a cell or organism a ds RNA molecule whose nucleotide sequence matches that of the gene to be inactivated • the RNA molecule hybridizes with the mRNA of target gene and directs its degradation • the degraded RNA are subsequently used by the cell to produce more ds RNA which directs the continued elimination of the target mRNA • these short RNA fragments can be passed on to progeny cells, RNAi can cause heritable change in gene expression • the RNA fragments can enter the nucleus and interact with the target gene itself, directing its packaging into chromatin

50. transgenic organisms 10_38_ES.cells.jpg ex: gene knockout mice