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Laboratory of Molecular Genetics, KNU Cell Culture.

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1 Laboratory of Molecular Genetics, KNU Cell Culture

2 Laboratory of Molecular Genetics, KNU Basics of Cell Culture

3 Laboratory of Molecular Genetics, KNU Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controll ed conditions. But in practice it refers to the cultu ring of cells derived from animal cells. Cell culture was first successfully undertaken by Ross Harrison in 1907 Roux in 1885 for the first time maintained embry onic chick cells in a cell culture Introduction

4 Laboratory of Molecular Genetics, KNU First development was the use of antibiotic s which inhibits the growth of contaminant s. Second was the use of trypsin to remove a dherent cells to subculture further from the culture vessel Third was the use of chemically defined cu lture medium. Major development’s in cell culture technology

5 Laboratory of Molecular Genetics, KNU Areas where cell culture technology is currentl y playing a major role. Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies Toxicity testing Study the effects of new drugs Cancer research Study the function of various chemicals, virus & radiatio n to convert normal cultured cells to cancerous cells Why is cell culture used for?

6 Laboratory of Molecular Genetics, KNU Contd…. Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. Genetic Engineering Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene

7 Laboratory of Molecular Genetics, KNU Choice of media depends o n the type of cell being cultu red Commonly used Medium ar e GMEM, EMEM,DMEM etc. Media is supplemented with antibiotics viz. penicillin, str eptomycin etc. Prepared media is filtered a nd incubated at 4 C Culture media

8 Laboratory of Molecular Genetics, KNU Laminar cabinet-Vertical are pr eferable Incubation facilities- Temperatu re of C for insect & 37 C f or mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubato rs set to cut out at approx C Refrigerators- Liquid media kep t at 4 C, enzymes (e.g. trypsin) & media components (e.g. gluta mine & serum) at -20 C Microscope- An inverted micro scope with 10x to 100x magnifi cation Tissue culture ware- Culture pla stic ware treated by polystyren e Basic equipments used in cell culture

9 Laboratory of Molecular Genetics, KNU If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen Swab all bottle tops & necks with 70% ethanol Flame all bottle necks & pipette by passing very quickly t hrough the hottest part of the flame Avoiding placing caps & pipettes down on the bench; pra ctice holding bottle tops with the little finger Work either left to right or vice versa, so that all material goes to one side, once finished Clean up spills immediately & always leave the work plac e neat & tidy Basic aseptic conditions

10 Laboratory of Molecular Genetics, KNU Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously until medium is thaw ed Centrifuge the vial for 10 mts at rpm at RT, wipe top of vial wi th 70% ethanol and discard the s upernatant Resuspend the cell pellet in 1 ml of complete medium with 20% FB S and transfer to properly labeled culture plate containing the appro priate amount of medium Check the cultures after 24 hrs to ensure that they are attached to t he plate Change medium as the colour ch anges, use 20% FBS until the ce lls are established Working with cryopreserved cells

11 Laboratory of Molecular Genetics, KNU Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration Dislodge the cells by trypsin-versene Dilute the cells with growth medium Transfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 mts at RT and remove the gr owth medium by aspiration Resuspend the cells in 1-2ml of freezing medium Transfer the cells to cryovials, incubate the cryovials at -80 C overnight Next day transfer the cryovials to Liquid nitrogen Freezing cells for storage

12 Laboratory of Molecular Genetics, KNU Cells are cultured as anchorage d ependent or independent Cell lines derived from normal tiss ues are considered as anchorage- dependent grows only on a suitabl e substrate e.g. tissue cells Suspension cells are anchorage-i ndependent e.g. blood cells Transformed cell lines either grow s as monolayer or as suspension Culturing of cells

13 Laboratory of Molecular Genetics, KNU Cells which are anchorage dependent Cells are washed with PBS (free of ca & mg ) so lution. Add enough trypsin/EDTA to cover the monola yer Incubate the plate at 37 C for 1-2 mts Tap the vessel from the sides to dislodge the c ells Add complete medium to dissociate and dislod ge the cells with the help of pipette which are remained to b e adherent Add complete medium depends on the subcult ure requirement either to 75 cm or 175 cm flask Adherent cells

14 Laboratory of Molecular Genetics, KNU Easier to passage as no need to detach them As the suspension cells reach to confluency Asceptically remove 1/3 rd of medium Replaced with the same amount of pre-warmed medium Suspension cells

15 Laboratory of Molecular Genetics, KNU Human cell lines -MCF-7 breast cancer HL 60 Leukemia HEK-293 Human embryonic kidney HeLa Henrietta lacks Primate cell lines Vero African green monkey kidney epithelial cells Cos-7 African green monkey kidney cells And others such as CHO from hamster, sf9 & sf21 from insect cells Common cell lines

16 Laboratory of Molecular Genetics, KNU Cell culture contaminants of t wo types Chemical-difficult to detect ca used by endotoxins, plasticize rs, metal ions or traces of disi nfectants that are invisible Biological-cause visible effect s on the culture they are myco plasma, yeast, bacteria or fun gus or also from cross-conta mination of cells from other ce ll lines Contaminant’s of cell culture

17 Laboratory of Molecular Genetics, KNU Cell viability is determined by staining the cells with trypan blue As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye Stain the cells with trypan dye and load to haemocyt ometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 100 total nu. of cells Cell viability

18 Laboratory of Molecular Genetics, KNU Cytotoxicity causes inhibition of cell growth Observed effect on the morphological alteration in the cell layer or cell shape Characteristics of abnormal morphology is the giant cells, multinucleated cells, a granular bumpy appear ance, vacuoles in the cytoplasm or nucleus Cytotoxicity is determined by substituting materials su ch as medium, serum, supplements flasks etc. at ati me Cell toxicity

19 Laboratory of Molecular Genetics, KNU Calcium phosphate precipitation DEAE-dextran (dimethylaminoethyl-dextran) Lipid mediated lipofection Electroporation Retroviral Infection Microinjection Transfection methods

20 Transfection of eukaryotic cells

21 Transfection What is Transfection? Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods. Methods: (just a few examples…) Method Application CaPO4, DEAE DNA Transfection Liposome Based DNA Transfection Polyamine Based DNA Transfection

22 Early Years Transfection = “transformation” + “infection” The infection of cells by the isolated nucleic acid from a virus, resulting in the production of a complete virus 1928: Frederick Griffith transforms Streptococcus pneumoniae 1944: Avery, Macleod and McCarty discover DNA is the transforming factor 1965: Vaheri and Pagano describe the DEAE-Dextran transfection technique 1964: Foldes and Trautner use the term transfection

23 Transfection vs. Transformation Transformation: genetically altering cells by transporting in foreign genetic material. Transfection: the process of transporting genetic material and/or macromolecules into eukaryotic cells through typically non-viral methods.

24 Purpose/uses of Transfection Study gene function Study protein expression Transfer DNA into embryonic stem cells

25 How it works Utilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of genetic materials or macromolecules.

26 Transfection method 1: DEAE- Dextran Facilitates DNA binding to cell membranes and entry via endocytosis DEAE-D associates with DNA  complex binds to cell surface Add chloroquine to transfection medium DMSO “shock” Add plasmid DNA to DEAE-Dextran medium Incubate cells with mixture…efficient transfection results in 25-75% death

27 Transfection method 1: DEAE- Dextran Major variants: number of cells, concentration of DNA and concentration of DEAE-Dextran Only method that cannot be used for stable transfections

28 Transfection method 3: liposome- mediated Use of cationic lipids –chemical and physical similarities to biological lipids –spontaneous formation of complexes with DNA, called lipoplexes High efficiency for in vitro transfections Can carry larger DNA than viruses Safer than viruses Low in vivo efficiency

29 How it works – Chemical and lipid methods Neutralize negative charges on DNA Chemical method: Calcium phosphate; creates precipitates that settle on cells and are taken in. Lipid or Polymer methods: interact with DNA, promotes binding to cells and uptake via endocytosis.

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31 Experimental method/process (chemical methods) HBS = HEPES-Buffered Saline

32 Transfection method 2: electroporation Use of high-voltage electric shocks to introduce DNA into cells Cell membranes: electrical capacitors unable to pass current Voltage results in temporary breakdown and formation of pores Harvest cells and resuspend in electroporation buffer Selection process for transfectant Add DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiled electroporate

33 Transfection method 2: electroporation Variants: amplitude and length of electric pulse Less affected by DNA concentration-linear correlation between amount of DNA present and amount taken up Can be used for stable transfections

34 How it works – Physical methods Electroporation: use of high voltage to deliver nucleic acids; pores are formed on cell membrane. Direct Microinjection: Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells. Biolistic Particle delivery: Uses high-velocity for delivery of nucleic acids and penetration of cell wall.

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36 Advantages/Disadvantages Advantages: Provides the ability to transfer in negatively charged molecules into cells with a negatively charged membrane. Liposome-mediated transport of DNA has high efficiency. Good for long- term studies requiring incorporation of genetic material into the chromosome. Disadvantages: Chemical Reagents: not useful for long-term studies. Transfection efficiency is dependent on cell health, DNA quality, DNA quantity, confluency (40-80%) Direct Microinjection and Biolistic Particle delivery is an expensive and at times a difficult method.

37 Laboratory of Molecular Genetics, KNU Exploring protein function 1) Where is it localized in the cell? 2) What is it doing in the cell? Approaches: a) Make antibodies - immunofluorescence Approaches: a) Reduce protein levels - RNA interference b) “Express” the protein in cells with a tag  Fuse to GFP b) Increase protein levels “over-express” c) “Express” mutant versions Transfection!!!!

38 Laboratory of Molecular Genetics, KNU Transfection = Introduction of DNA into mammalian cells Gene is transcribed and translated into protein = “expressed”

39 Laboratory of Molecular Genetics, KNU Direct introduction of the DNA Electroporation - electric field temporarily disrupts plasma membrane Biolistics (gene gun)- fire DNA coated particles into cell Microinjection

40 Laboratory of Molecular Genetics, KNU Infection: Use recombinant viruses to deliver DNA Retroviruses Adenoviruses Virally-mediated introduction of the DNA

41 Laboratory of Molecular Genetics, KNU Positively charged carrier molecules are mixed with the DNA and added to cell culture media: Calcium Phosphate DEAE Dextran liposomes micelles Carrier-DNA complexes bind to plasma membrane and are taken up Carrier-mediated introduction of the DNA

42 Laboratory of Molecular Genetics, KNU Types of Transfection Transient: Expression assayed hours post transfection Stable: Integration of the transfected DNA into the cell genome - selectable marker like neomycin resistance required “stably transfected” cell line

43 Laboratory of Molecular Genetics, KNU DNA “expression” vector transfected: pCMV/GFP CMV Promoter Insert gene in here Polyadenylation site SV40 Promoter Neomycin resistance Polyadenylation site pUC Bacterial origin of replication Ampicillin resistance For expression in cells To generate stable cell line For amplification of the plasmid in bacteria GFP

44 Laboratory of Molecular Genetics, KNU PROTEIN X PROTEIN Y GFP Z PROTEIN Three ways to make Green fluorescent protein “GFP” fusion constructs:

45 Laboratory of Molecular Genetics, KNU EXPERIMENT: Transfect unknown GFP fusion protein Protein X, Y or Z Visualize GFP protein fluorescence by fluorescence microscopy in living cells Counter-stain with known marker to compare localization patterns in living cells = “vital stain”

46 Laboratory of Molecular Genetics, KNU Some Cellular Organelles

47 Laboratory of Molecular Genetics, KNU Compartments/organelles examined Protein sequences sufficient for localization Vital stains Secretory Pathway: Endoplasmic Reticulum Golgi Complex Endocytotic Pathway: Endosomes Mitochondria Nuclei

48 Laboratory of Molecular Genetics, KNU Transport through nuclear pore signal = basic amino acid stretches example: P-P-K-K-K-R-K-V Nucleus

49 Laboratory of Molecular Genetics, KNU Import of proteins into nucleus through nuclear pore

50 Laboratory of Molecular Genetics, KNU Nuclear Stain: Hoechst binds DNA

51 Laboratory of Molecular Genetics, KNU Transmembrane transport signal Example: H 2 N-M-L-S-L-R-Q-S-I-R-F-F-K- P-A-A-T-R-T-L-C-S-S-R-Y-L-L Mitochondria

52 Laboratory of Molecular Genetics, KNU Protein being transported across mitochondrial membranes

53 Laboratory of Molecular Genetics, KNU Mitochondrial dye = MitoTracker Red Non-fluorescent until oxidized Accumulates in mitochondria and oxidized Diffuses through membranes Mitotracker DNA

54 Laboratory of Molecular Genetics, KNU nuclear envelope endoplasmic reticulum lysosome early endosome late endosome Golgi apparatus cis Golgi network trans Golgi network Golgi stack CYTOSOL plasma membrane Cellular components of the secretory and endocytic pathways

55 Laboratory of Molecular Genetics, KNU Entry into E.R.: Transmembrane transport signal = hydrophobic amino acid stretches Example:H 2 N-M-M-S-F-V-S-L-L-V-G-I-L- F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q Retention in E.R. lumen: Signal = K-D-E-L-COOH Endoplasmic Reticulum at amino terminus at carboxy terminus

56 Laboratory of Molecular Genetics, KNU ER-Tracker Blue-White Live bovine pulmonary artery endothelial cells Endoplasmic Reticulum marker

57 Laboratory of Molecular Genetics, KNU Mitotracker Red and ER- blue/white

58 Laboratory of Molecular Genetics, KNU Golgi nucleus From the ER, secreted and membrane proteins move to the Golgi, a series of membrane-bound compartments found near the nucleus

59 Laboratory of Molecular Genetics, KNU BODIPY-TR ceramide Golgi marker Ceramide = lipid When metabolized, concentrates in the Golgi Red fluorophore

60 Laboratory of Molecular Genetics, KNU Golgi (ceramide) DNA (Hoechst) Cultured Epithelial Cells

61 Laboratory of Molecular Genetics, KNU Golgi (ceramide) Lysosomes (LysoTracker) DNA (Hoechst) MDCK Cells Madin-Darby Canine Kidney Polarized Epithelial Cells Molecular Probes, Inc.

62 Laboratory of Molecular Genetics, KNU Rhodamine transferrin Does the fluorescent green protein co-localize?

63 Laboratory of Molecular Genetics, KNU Immunofluorescence / confocal microscopy 원리

64 Laboratory of Molecular Genetics, KNU Immunofluorescence / confocal microscopy B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue: 1. wash cells 1x cold RPMI (no wash for cryostat sections). 2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. ** 3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice. 4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA. 5. wash 1x PBS/BSA. 6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA. 7. wash 1x PBS/BSA. 8. 1° diluted in PBS/BSA 60 min RT; 100 l per tube or section. 9. wash 3x PBS/BSA. 10. Block 15 min 5% NGS in PBS/BSA. 11. wash 1x PBS/BSA ° diluted in PBS/BSA 30 min RT; 100 l per tube or section. 13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass just put two-three drops in each chamber after wash. Immunofluorescence / confocal microscopy 실험방법

65 High-throughput loss-of-function screens using RNAi cell microarrays.

66 An example of a high-density, high-content RNAi cell-microarray screen in cultured D. melanogaster Kc167 cells.

67 Laboratory of Molecular Genetics, KNU Experimental Animals

68 Laboratory of Molecular Genetics, KNU Model systems E. coli - easy and inexpensive to maintain - 사람 장내 서식, 배설물의 주성분 - 해로운 박테리아의 생장 저해 Pathogenic 은 sickness, death 유발 - 돌연변이 잘 유발 → Metabolism 연구에 이용 Eukaryote 에서의 과정 일어나지 않음

69 Laboratory of Molecular Genetics, KNU Yeast - Eukaryote 와 prokaryote 사이의 차이점 연구에 이용 - Genetic study easy - haploid, single-celled - Grow very rapidly, inexpensive - 2 가지 종류 사용 → Saccaromyces cerevisiae; budding → Schizosaccaromyces pombe; fission

70 Laboratory of Molecular Genetics, KNU Cloning the yeast origin of replication - yeast genome cut restriction enzyme - fragments clone into plasmid vector - recombinant plasmids growth and select - yeast survive in media lacking leucine - selected yeast cells contain leu2+ gene

71 Laboratory of Molecular Genetics, KNU Nematoda worm - Small and easy to keep - Three days to develop Multicellular organism Caenorhabditis elegans Fruit fly Identification of mutant → mutation 특징 확인이 쉬움 - 짧은 시간에 많은 자손 생산 - 알에서 성체가 되기까지 12 일 소요 - Drosophila melanogaster

72 Laboratory of Molecular Genetics, KNU Zebrafish - Reproduction rapidly and high number - vertebrate - 발생 과정이 사람과 유사 - 알이 투명하여 발달 확인 가능 Danio rerio Amphibians - 알의 크기가 큼 → 발생학 연구에 이용 년 슈페만 형성체 발견 - Xenopus leavis

73 Laboratory of Molecular Genetics, KNU Chicken 배 발생 연구에서 알 사용됨 ; 진화상 인간과 유사 - 알이 크고, 분화 관찰에 좋음 - Gallus gallus Mouse - vertebrate, mammals Human 과 더욱 가까움 ; 생리학, 발생학적으로 유사 - Expensive to maintain, Reproduce slowly Knock out mouse 이용 → 특정 gene 의 기능 확인

74 Laboratory of Molecular Genetics, KNU Human cell culture Human blood 나 tissue 에서 분리 Primary culture ; derived directly from living tissue - Grow for a short period time and stop - 세포 분열 횟수 제한 Plants - Grow slowly, long generation time - 세포벽 때문에 형질전환이 어려움 - Large genome - Transposon 연구에 이용 ; corn - Arabidopsis thaliana


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