Manipulating genes and cells (Kap. 10)

Transcription

1 Manipulating genes and cells (Kap. 10) restriction enzymes and agarose gel electrophoresis DNA sequencing nucleic acid hybridization techniques genomic and cdna libraries cloning of DNA PCR and PCR applications isolating cells and growing them in culture protein expression in recombinant cell lines genetically altered animals and plants

2 Experimenting with DNA Traditional crossbreeding Genetic engeneering Transfer of many genes Genes are not known Large changes in protein expression Within species Transfer of a single or a few gene Genes are known Small changes in protein expression Between species

3 DNA

4 The DNA-helix In 1953, based on X-ray diffraction images taken by Rosalind Franklin and the information that the bases were paired, James D. Watson and Francis Crick suggested the DNA structure in the journal Nature. Experimental evidence for Watson and Crick's model were published in a series of five articles in the same issue of Nature.

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6 Restriction enzymes, molecular scissors from bacteria EcoR1 bound to DNA

7 Restriction endonucleases cleave DNA at specific nucleotide sequences

8 The Nobel Prize in Physiology or Medicine 1978 "for the discovery of restriction enzymes and their application to problems of molecular genetics" Werner Arber Daniel Nathans Hamilton O. Smith

9 Gel electrophoresis to separate DNA molecules

10 Agarose gel electrophoresis

11 DNA sequencing (1) Dideoxy method

12 DNA sequencing (2)

13 DNA sequencing (3)

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15 DNA sequencing using fluorescent dies and a laser detector

16 DNA sequencer (capillary electrophoresis)

17 Celera Genomics (Craig Venter) and the Human Genome Sequencing Consortium (Francis Collins) sequenced the human genome Francis Collins (NIH NHGRI) Craig Venter, Celera Genomics

18 Strategies for sequencing complex genomes Clone-by-clone shotgun sequencing Whole-genome shotgun sequencing

19 Important genome sequencing papers Mouse Nature. 420: (5 December 2002) Human Nature. 409: (15 February 2001) Arabidopsis - First Plant Sequenced Nature 408: (14 December 2000) Fruit Fly Science. (24 March 2000) 287: Roundworm C. elegans - First Mutlicellular Eukaryote Sequenced Science. (11 December 1998) 282: Bacteria - E. coli Science. 277: (5 September 1997) Yeast Science. (25 October 1996) 274: 546, Bacteria - H. influenzae - First Free-living Organism to be Sequenced Science. (28 July 1995) 269:

20 There are more than 600 genomes completey sequenced in 2007 See GOLD (genome online databases at

21 The future of DNA sequencing In May 2007, James D. Watson, the co-discoverer of the molecular structure of DNA, became the first person to receive his own complete personal genome sequenced. The cost was less than $1 million (0.03 cents per base). It took less than two months. It is realistic to expect that within the next ten years, rapid low-cost sequencing of human genomes will become a reality. Pharmacogenomics Genotyping

22 Hybridization of DNA

23 Southern blotting (DNA) Northern blotting (mrna) Western blotting (protein)

24 E. Southern, "Detection of specific sequences among DNA fragments separated by gelelectrophoresis," J Mol Biol, 98:503, 1975.

25 A Northern blot shows amount and size of mrna of 1 gene present in different tissues

26 DNA microarrays (DNA chips) look at many genes (whole transcriptomes) at once glas or silicium chip each dot represents DNA of one gene spotted on the chip and hybridized with the 2 probes -> identify up- or downregulated genes in different samples

27 Genes (DNA) can be located on chromosomes by fluorescence in situ hybridization (FISH) 6 different DNA probes 2 dots on the maternal, 2 on the paternal chromosome 5 (chromosomes have already replicated their DNA)

28 mrna can be located on cells by in situ hybridization Radioactively labeled probe, exposure on X-ray film Non-radioactive labeled probe, visible precipitating reaction product

29 Multi-labeling techniques different coloured enzymatic reaction products: Different fluorochromes:

30 Ligation of DNA to form recombinant DNA molecules in vitro

31 Some bacteria can efficiently take up DNA from their surrounding introduction of DNA into bacteria: transformation introduction of DNA into eukaryotic cells: transfection

32 A typical plasmid cloning vector

33 DNA cloning DNA ligase inserts a DNA fragment into a bacterial plasmid DNA plasmid is introduced into a bacterium and replicates

34 Construction of a genomic library to isolate human genes by DNA cloning

35 Colony hybridization: identification of bacterial colonies containing a particular DNA

36 Synthesis of cdna (reverse transcriptase) Many essential molecular biology techniques (working with restriction enzymes, ligations) do not work with single-stranded mrna mrna is rather instable and rapidly degraded by ubiqitary RNAses Therefore, the sequence information from a mrna molecule is first transcribed into a more stable and clonable DNA copy (cdna)

37 Genomic versus cdna clones

38 PCR (polymerase chain reaction) amplifies DNA sequences

39 The Nobel Prize in Chemistry 1993 Michael Smith Canada, for his fundamental contributions to the establishment of oligonucleotide-based, sitedirected mutagenesis and its development for protein studies Kary B. Mullis USA, for his invention of the polymerase chain reaction (PCR) method

40 PCR machine

41 Obtaining genomic or cdna clones by PCR

42 PCR can be used to detect viral infections in a blood sample

43 Real-time or quantitative PCR Often using SYBRE-Green, a dye that emits fluorescent light only when bound to double-stranded DNA produced in the PCR-reaction tube

44 Recombinant protein expression in cell lines New recombinant plasmids are spliced together Large amounts of protein are produced in cell culture

45 An expression vector (plasmid DNA) MCS (multiple cloning site) Strong eucaryotic promoter from the cytomegalo virus Resistence gene for E. coli Resistence gene for mammalian cell lines

46 From gene to protein and from protein to gene

47 Fluorescence-activated cell sorting (FACS) to isolate specific types of cells

48 FACS

49 Cells in culture Fibroblast secrete collagen Muscle cells contract Nerve cells make synapses Epithelial cells make sheets

50 HeLa cells (cervical cancer from Henrietta Lacks) Nomarsky Phase contrast

51 CHO-K1 (chinese hamster ovary) cell line

52 A cell culture lab

53 Working in cell culture sterile, sterile, sterile! 12-wells 6-wells 24-wells 96-wells 1536-wells

54 Reporter genes can determine the pattern of a gene s expression When is the gene expressed? Where is the gene expressed? How much?

55 Green fluorescent protein (GFP)

56 Bioluminescence reaction in aequorea victoria

57 Green fluorescent protein (GFP) can identify specific cells in a living animal About 20 neurons in a Drosophila embryo express GFP under the control of a neuronal fly promoter

58 GFP expressed in the eyes of Drosophila

59 A cell-membrane protein fused to GFP

60 Fluorescent proteins

61 Spectra of fluorescent proteins

62 β-galactosidase as a reporter gene Section of a fly brain

63 Genetically engineered organisms

64 Gene addition: e.g. GFP as a marker in transgenic animals

65 Creating a Knockout mouse using embryonic stem cells (ES cells) to selectively delete a single gene/protein The phenotype of this KOmouse can give a hint to the physiological function of this protein in the wild-type organism

66 Antisense RNA can generate dominant negative mutations

67 RNA interference (RNAi) knock-down

68 Transgenic plants can be made with an Agrobacterium

69 A tobacco plant expressing GFP

70 Bio-pharming (transgenic plants) Inserting genes that instruct a plant to manufacture pharmaceutical compounds: plants as drug-producing bioreactors.

71 Golden rice Rice expressing 3 transgenes responsible for provitamin-a (beta-carotene) synthesis

72 Gene-pharming (transgenic animals) mostly used to make human proteins that have medicinal value. The protein is secreted into the animal's milk, eggs or blood, and then collected and purified.

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74 Genetic engineering and ethical issues therapeutical cloning PDS (pre-implantation genetic diagnosis and selection) germline cloning