Ectopic Gene Expression in Mammalian Cells

Transcription

1 Ectopic Gene Expression in Mammalian Cells Dr. Stefan Stein AG Grez Methodenseminar BCII Praktikum am Georg-Speyer-Haus &

2 Ectopic Gene Expression in Mammalian Cells Definition: ectopic Etymology: Greek ektopos out of place, from ex- out + topos place occurring in an abnormal position or in an unusual manner or form 2

3 Ectopic Gene Expression in Mammalian Cells Plasmid DNA / Virus Transfer (transient / stable) nucleus RNA Analysis, Protein, Reporter Gene Expression PCR (genomic DNA) 3

4 The TATA-box - starting site for assembling of general transkription factors TATA binding protein - consensus sequenz: 5'-TATAAA-3 - binding site for TATA-Box binding protein (TBP) Protein-DNA interaction - 25 nucleotides downstream: transcriptional starting site (TSS) = binding site for RNA polymerase DNA 4

5 Simple Scheme T-Factor T-Factor Promotor inactive Promotor active and Real Life Promotor??? more or less active C. Brendel 5

6 Transcriptosom 6

7 Gene Expression Vectors 7

8 Gene Expression Vectors: Promotor Types (non-coding seq.) (e.g.: CMV) (coding cdnas) (lck) (T-cell, B-cell,...) (tet) CAAT box signals the binding site for RNA transcription factors; consensus sequence: GGNCAATCT 8

9 Reporter Genes - The reporter gene must not encode a protein activity that is similar to one already present in the target cell - The protein assay needs to be sensitive over several orders of magnitude, reproducible, and easy to perform. In this regard, it is also helpful if the assay is time and cost-effective (screening) - The reporter protein function must not interfere with host cellular processes in a way that will alter intracellular signaling pathways or metabolic rates. 9

10 Selecetion/detection of transfected/transduced cells 1. Selection marker: neomycin resistance (aminoglycosid) kanamycin, puromycin transfection selection experiment (1-2 weeks) 2. Fluorescent protein: GFP, YFP, RFP,... transduction cell sorting after 3-4 days: experiment 3. PCR 10

11 Reporter Genes: GFP tokyo.ac.jp/bioresource/shimada/images/insects/bombyx_mori/b.mori- adult/gfp-transgenic.jpg -%20GFP%20Mouse%20-%20800X600.jpg 11

12 Fluorescent Proteins: Spectrum of Colours 12

13 Reporter Genes: Luciferase 13

14 Example of a Plasmid for Expression in Mammalian Cells MCS - Invitrogen - 14

15 How to get in?? plasmid DNA / virus transfer (transient / stable) RNA Analysis Reporter Gene Expression 15

16 Methods for transfer of genetic material into mammalian cells - CaPO 4 transfection - lipofection - electroporation - mikroinjection - viral transfer - non-viral 16

17 Transfection using Calcium Phosphate 293T 17

18 Transfection using Calcium Phosphate: Procedure 18

19 Transfection of 293T Cells - + excitation (Fluorescence microscope) 19

20 Lipofection Lipofektion: 20

21 From liposomes to nanoparticals Targeted delivery of drugs! 21

22 Electroporation - Stromspannung (Volt) - Impulsdauer - Temperatur (Zellen auf Eis) - DNA Konzentration - Ionenzusammensetzung des Elektroporationsmediums 22

23 Electroporation chamber Cave: 40-90% cell death! 23

24 Microinjection 24

25 Microinjection DNA (transgenic animals) RNA (e. g.: in Xenopus eggs) RNAi (knock-down) proteins nuclei! (cloning) sperm cells (in vitro fertilisation) 25

26 Retroviruses RNA viruses infection of cells via receptor mediated endocytosis RNA genome is transcribed into DNA (reverse transcription) integrate into host genome (provirus) efficient tool for (stable) introduction of genetic material in mammalian cells commonly used vector system for somatic gene therapy 26

27 Structure of a Retrovirus (2 RNA strands) LTRs: long terminal repeats (contain enhancer/promotor region for control of expression) Ψ + (psi): packaging signal 27

28 The Replication Cycle of Retroviruses virus translation: protein of desire (e.g. GFP) retro_lifecycle.mov 28

29 Production of Retroviral Particles Wildtyp virus (replication competent) LTR Ψ gag pol env LTR Transfer construct (replication incompetent) LTR Ψ gene X LTR Ψ gag pol Packing cell line (gag-pol/env in trans) Ψ env 29

30 Production of Retroviral Particles Transduktion gp91 30

31 Somatic Gene Therapy for a Blood Disorder Reinfusion of genetically modified cells G-CSF Mobilisation Leukapheresis Quality Control Retroviral mediated Gene Transfer Isolation of CD34 + Cells U3 R U5 gp91 phox U3 R U5 Blood Stem Cells (CD34+ cells) Gene Transfer Vector (SF71gp91phox) 31

32 Percentage of transduced and functional cells in peripheral blood of patient % ce ells containing transg gene days after transplantation % functional cells 32

33 Insertional Mutagenesis and Gene Silencing A gp91-phox GOI B Enhancer /Promoter active Enhancer /Promoter inactive 33

34 Vectors Used in Gene Therapy Clinical Trials September 2001: 593 trials June 2011: 1714 trials 34

35 Stable Expression via Lentiviral Vectors U3 gag RRE Ψ R U5 R U5 sd sa cppt SFFV Flag-NLS Peptide IRES egfp U3 lentiviral expression vector (SiEW) (SFFV: promotor/enhancer element from spleen focus forming virus LTR) SSC Kasumi-1 - mock mock +anex.001 R FL-1 (egfp) surface markers CD11b, CD14, CD34,... FACS sorting, growth curve cell cycle distribution (Draq5) 35