Molecular basis of agrobacterium mediated gene transfer into mammalian cells 2

Chapter 2. Materials and Methods 2.1. Bacterial strains, plasmids, primers, media and antibiotics Bacterial strains and plasmids used in this study are listed in Table 2.1. Preparations of media used in this study for the growth of bacterial strains were formulated as listed in Table 2.2. For long-term storage, the bacteria were kept in LB with 50% glycerol at –80°C. Escherichia coli strains were grown at 37°C in LB (Sambrook et al., 1989) and Agrobacterium tumefaciens strains were grown at 28°C in MG/L, AB or IB media (Cangelosi et al., 1991) supplemented with the appropriate antibiotics when necessary. Rhizobium meliloti (Finan et al., 1986) cells were grown in LB/MC medium supplemented with 10 µg/ml tetracycline when necessary (Glazebrook and Walker, 1991). Plasmid DNA was introduced into A. tumefaciens strains or Rhizobium meliloti by electroporation (Ditta, 1980; Glazebrook, 1991). The preparation and concentration of antibiotics and other solutions used in this study are listed in Table 2.3. 2.2. Cell culture and culture medium Mammalian and plant cell lines used in this study are listed in Table 2.4. 2.2.1. Mammalian cell culture 2.2.1.1. Dulbecco’s Modified Eagle’s Medium (DMEM) DMEM was used to culture mammalian cells such as human adenocarcinoma cell line (HeLa), human embryonic kidney (HEK) EcoPack2-293 cells and PT67 mice 46 Table 2.1. Bacterial strains and plasmids Bacterial strain or plasmid Relevant characteristic(s) Source or reference DH5α EndA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 ∆(argF-lacZYA)U169 φ80dlacZ ∆Μ15 Bethesda Research Laboratories MT607 Pro-82 thi-1 hsdR17 supE44 end44 endA1 recA56 Finan et al, 1986 MT616 MT607(pKR600), mobilizer Finan et al, 1986 S17.1(λpir) λpir lysogen, recA, thi, pro, hsdR-M+, RP4:2Tc:Mu:KmR , Tn7, TpR , SmR De Lorenzo et al., 1993 Wild type, nopaline-type pTiC58 plasmid Laboratory collection A348 A136 (pTiA6NC) (octopine-type) Garfinkel et al., 1981 A136 C58 cured of pTiC58, RfR, NaIR Watson et al, 1975 Mx243 A136 containing pTiA6 virB1::Tn3-HoHo1; virB1- Stachel and Nester, 1986 Mx226 A136 containing pTiA6 virA::Tn3-HoHo1; virA- Stachel and Nester, 1986 Mx306 A136 containing pTiA6 virD1::Tn3-HoHo1; virD- Stachel and Nester, 1986 Mx358 A136 containing pTiA6 virE1::Tn3-HoHo1; virE- Stachel and Nester, 1986 Mx363 A136 containing pTiA6 virG1::Tn3-HoHo1; virG- Stachel and Nester, 1986 CGI1 Derivative of C58 in which aopB was disrupted by the GFP-tagged mini-Tn5 transposon This study A6340 A6007 chvG340::TnphoA; chvG- Cangelosi et al., 1991 A6880 A6007 chvH340::TnphoA; chvH- Cangelosi et al., 1991 AG6 A348 catalase::mini-Tn5; katA- Xu and Pan, 2000 Strains Escherichia coli Agrobacterium tumefaciens C58 47 GMI9023 C58 cured of pTiC58 and pAtC58 Truchet et al., 1984 A136::Tn5 A136 derivative with mini-Tn5 insertion This study A348::Tn5 A348 derivative with mini-Tn5 insertion This study GMI::Tn5 GMI9023 derivative with mini-Tn5 insertion This study Same as SU47 wild type Charles and Nester, 1993 Rhizobium meliloti RCR2011 Plasmids pTZ19R Cloning vector, ColE1 oriV bla, AmpR R US Biochemical pLEGFP-C1 Expression vector, ColE1 oriV, Amp Clontech pSW172 Broad-host-range IncP plasmid containing Plac and downstream polylinker sequence, TcR Chen and Winans, 1991 pBSL202 Plasmid harboring mini-Tn5 transposon, GmR, AmpR Suarez et al., 1997 pCB301 Broad-host-range plasmid derived from pBIN19, KmR Xiang et al., 1999 pQM45 pCB301 carrying a kb neoegfp fragment from pNEOEGFP between T-borders This study pQM49 pSW172 carrying a 0.8 kb gfp fragment from pAG408, TcR This study pQM52 pCB301 harboring pLEGFPC-1 between Tborders, AmpR This study pQM54 pSW172 digested with StuI and ligated to pLEGFPC-1digested with XmnI, TcR This study pQM61 pBSL202 harboring pLEGFPC-1 between its insertion sequences, AmpR This study 48 Table 2.2. Media preparation Preparationa, b Reference Tryptone, 10 g; yeast extract, g; NaCl, 10 Sambrook et al., g; pH 7.5 1989 Tryptone, 20 g; yeast extract, g; NaCl, 0.5 Sambrook et al., g; 10 ml of 250 mM KCl; pH 7.0,sterilize 1989 Media or solutions LB (Luria broth) SOB by autoclaving and add 5ml of filtersterilized M MgCl2. TB 10 mM PIPS, 55 mM MnCl2, 15 mM CaCl2, 250mM KCl; MG/L LB, 500 ml; mannitol, 10 g; g sodium Cangelosi et al., glutamate, 2.32; KH2PO4, 0.5 g; NaCl, 0.2 1991 g; MgSO4. 7H2O, 0.2 g; biotin, µg; pH 7.0. AB (Minimal 20 × AB salts, 50 ml; 20 × AB buffer, 50 Cangelosi et al., medium) ml; 0.5% glucose 900 ml (autoclaved 1991 separately before mix together). IB (Induction 20 × AB salts, 50 ml; 20 × AB buffer, ml; Cangelosi et al., Medium) 0.5 M MES (pH 5.5), ml; 30% glucose, 1991 60 ml (autoclaved separately before mix together). 20 × AB salts 20 × AB buffer NH4Cl, 20 g; MgSO4. 7H2O, g; KCl, g; Cangelosi et al., CaCl2, 0.2 g; Fe SO4. 7H2O, 50 mg. 1991 K2HPO4, 60 g; NaH2PO4, 23 g; pH7.0. Cangelosi et al., 49 1991 0.5 M MES MES, 97.6 g; pH5.5. Cangelosi et al., 1991 1000 × ASc 14.6 mg/ml AS in DMSO. Sambrook et al., 1989 MS medium a Murashige and Skoog salts and vitamin mixture, 4.42g; sucrose, 30g; 2, 4-D (0.1 mg/ml), 2ml Murashige and Skoog, 1962 Preparation for liter, and sterilized by autoclaving; b For solid media, 1.5% agar was added; c no autoclaving is necessary. 50 Table 2.3. Antibiotics and other stock solutions used in this study Name Preparations Stock Concentration (Con.) (mg/ml) Working Con. in E. coli (µg/ml) Working Con. in A. umefaciens (µg/ml) ampicillin (Amp) Dissolved in dH2O, filter sterilized Same as above 100 100 -- kanamycin (Km) Carbenicillin Same as above (Cb) gentamycin Same as above (Gm) tetracycline (Tc) Dissolved in absolute ethanol chloramphenical Same as above (Chl) 100 50 100 100 100 100 50 10 50 10 34 17 -- Acetosyringone Dissolved in dimethyl sulfoxide. 100 mM -- 100 µm IPTG Dissolved in dH2O, filter sterilized 24 24 24 X-Gal Dissolved in dimethyl sulfoxide 20 20 20 Proteinase K Dissolved in dH2O 20 50 50 RNase Dissolved in dH2O 10 20 20 51 Table 2.4. Mammalian and plant cell lines cell line Relevant characteristic(s) Source or reference BY-2 Nicotiana tabacum L. cv. Bright Yellow callus suspension cells Laboratory collection HeLa human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 Laboratory collection EcoPack2-293 An ecotropic, HEK 293-based packaging cell line ideal for transiently or stably producing virus capable of infecting mouse and rat cells. Clontech RETROPACKTM PT67 A dualtropic, NIH 3T3-based packaging cell line ideal for stably producing virus capable of infecting a broad range of mammalian cell types. Clontech 52 cells (Clontech). It contains 25 mM HEPES, 4mM L-glutamine, 4.5g/l glucose, 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100µg/ml streptomycin. For the culture of HEK-293 and PT67 cells, DMEM was supplemented with 1nM sodium pyruvate for optimal growth. All tissue culture reagents used were obtained from Sigma. 2.2.1.2. Mammalian cell culture and subculture HeLa, EcoPack2-293 and PT67 cells were grown in DMEM at 37°C in a % (v/v) CO2 incubator. Cells were maintained in 75 cm2 flasks and subcultured at least once every days by trypsin/EDTA treatment and at a dilution of 1:4 in fresh medium. 2.2.2. Plant cell culture and subculture Tobacco BY2 callus (N. tabacum Bright Yellow 2) cells were maintained on solid Murashige and Skoog’s medium (MS; Murashige and Skoog, 1962) supplemented with 3% sucrose and 0.2 mg/ml 2,4-D. For using in A. tumefaciens mediated transformation and attachment assay, the BY2 cells were grown in liquid MS medium at room temperature with shaking at 100 rpm and were subcultured every week with a % inoculum. 2.3. DNA manipulations 2.3.1. Plasmid DNA preparation Plasmid DNA was prepared following the method described previously with some modifications (Sambrook et al., 1989). Briefly, E. coli cells from ml of overnight culture were collected by centrifugation at 10, 000 rpm (Eppendorf 5417C) 53 for min. The cell pellet was resuspended in 100 µl of ice-cold solution I (50mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) thoroughly by vigorous vortex. Then, 200 µl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and the contents were mixed by inverting gently for 4-6 times. After the addition of 150 µl of Solution III (3 M potassium, M acetate), the mixture was inverted for 4-6 times to disperse Solution III through the viscous bacterial lysate. The lysate was extracted with equal volume of chloroform once by centrifuging at 14, 000 rpm (Eppendorf 5417C) for min. The supernatant was then transferred to a clean eppendorf tube. To precipitate the plasmid DNA, volumes of ethanol was added and the mixture were centrifuged as above. The DNA pellet was washed once with 70% ethanol and dried in a vacuum concentrator. The extracted plasmid DNA was dissolved in 20 µl of sterile water and stored at -20 °C, ready for subsequent use after thawing. 2.3.2. Genomic DNA preparation from Agrobacterium Genomic DNA of Agrobacterium was prepared according to Charles and Nester (Charles and Nester, 1993). Cells from 100 ml of overnight culture were harvested by centrifugation at 3000 rpm for min. The cells were washed once with ml of TES (10 mM Tris-HCl, 25 mM EDTA, 150 mM NaCl, pH 8.0) and resuspended in ml of TE buffer (10 mM Tris-HCl, 25 mM EDTA, pH 8.0). To lyse the cells, 500 µl of M NaCl, 500 µl of proteinase K (5 mg/ml), and 500 µl of 10% SDS were add to the cell suspension and then incubated at 68 °C for 30 min. The lysate was extracted once with 1:1 phenol-chloroform and then chloroform alone. To precipitate genomic DNA, 7.5 M ammonium acetate was added to the final concentration of M and then volumes of ethanol were added. The DNA pellet was washed once with 70% ethanol 54 and vacuum dried. Genomic DNA was dissolved in 500 µl of distilled water and stored at °C. 2.3.3. DNA digestion and ligation DNA digestion and ligation were conducted following the instructions of the manufacturers supplying the enzymes. Digestion reaction systems comprised of buffer, enzyme, DNA and water, and incubated at 37 °C for hour to overnight as required. For vectors digested with a single enzyme, dephosphorylation was carried out by adding 0.5 µl of shrimp alkaline phosphatase into the digestion mixture. Digested vectors and gene fragments used for ligation were cleaned using QIAGEN gel extraction kit. Ligation was carried out by incubating the mixture of T4 DNA ligase, vector DNA, insertion DNA, ligase buffer and water at room temperature for h or overnight. 2.3.4. Polymerase chain reaction (PCR) Polymerase chain reaction was carried out using a thermocycle (Applied Biosystem) in a thin wall PCR tube with a volume of 200 µl. The reaction mixture usually contained the following components in a final volume of 50 µl: 55 10 × PCR buffer (without MgCl2) µl 25 mM MgCl2 µl Primer (10 pmol/µl) µl Primer (10 pmol/µl) µl dNTPs (10 mM each) µl Template DNA 20-100 ng Taq DNA polymerase µl (1 unit) Add distilled water to a final volume of 50 µl The PCR was run using the following program: cycle 95 °C for 30 cycles 95 °C for 30 seconds Annealing at (Tm-5) °C for 30 seconds Extension at 72 °C for per kb cycle 72 °C for 10 56 2.3.5. DNA gel electrophoresis and purification DNA fragments were electrophoresized in an × TAE (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) agarose gel along with a standard DNA marker (Fermentas). Digested DNA vectors and fragments from genomic DNA or PCR products to be used for ligation and transformation reaction were usually purified with QIAquick Gel Extraction Kit (QIAGEN) following the instructions provided by the manufacturer. Briefly, DNA was separated in an 1% agarose gel. The gel slice containing the desired DNA bands were excised and transferred to a pre-weighted eppendorf tube. Then gel volumes (100 mg gel ≈ 100 µl) of buffer QG were added and the tube was incubated in an 55 °C waterbath for 5-10 to dissolve the gel completely. For DNA fragments larger than kb or smaller than 500 bp, gel volume of isopropanol was added. The mixture was transferred to a QIAquick spin column in an 2-ml collection tube. The binding of DNA to the column was achieved by centrifugation for at 14, 000 rpm (Eppendorf 5417C). The column was then washed once with 750 µl of buffer PE with one additional centrifugation to remove residual ethanol. The column was placed into a clean 1.5-ml centrifuge tube. To elute DNA, 50 µl of sterile water was applied to the center of the column membrane and the column was centrifuged at 14, 000 rpm (Eppendorf 5417C) for min. 2.3.6. Preparation of competent cells E. coli DH5α was routinely used as the host for cloning experiments unless otherwise specified. High efficient competent cells were prepared as described previously (Inoue et al, 1990). E. coli cells were streaked from frozen stock and cultured overnight on an LB plate at 37 °C. Then several colonies were picked and inoculated into 100 ml of SOB medium in a 1-liter conical flask. The cells were 57 cultured at room temperature (about 19°C) with vigorous shaking (250 rpm) to an OD600 of 0.5-0.7. The cells were chilled on ice for 10 before they were collected by centrifugation at 2600 rpm (Eppendorf 5810R) for at °C. The cell pellets were resuspended in 30 ml of ice-cold TB buffer (10 mM PIPES, 55 mM MnCl2, 15 mM CaCl2, 250mM KCl, pH 6.7; all components except MnCl2 were dissolved and autoclaved; 1M MnCl2 solution was filter-sterilized and added to make TB buffer; store at °C) and then incubated on ice for 10 min. Cells were collected by centrifugation as above and resuspended in ml of ice-cold TB buffer. Thereafter, DMSO was added to a final concentration of 7% and the cell suspension was aliquoted into pre-cooled sterile eppendorf tubes at 100 µl each. The competent cells were kept at -80 °C until needed. 2.3.7. Transformation of E. coli A plasmid or a ligation reaction product was introduced into E. coli by transformation for amplification or screening (Sambrook et al, 1989). A frozen competent cell (100 µl) was thawed on ice. Plasmid (50-100 ng in 10µl or less) or ligation product (10 µl) was added and the contents of the tube were mixed by gently tapping the tube a few times. The tube was then incubated on ice for 30 minutes. The mixture of cells and DNA were heat-shocked at 42 °C for 90 seconds. After chilling the cells on ice for min, 900 µl of fresh LB medium were added. The cultures were incubated at 37 °C for 30 with agitation. The cells were collected and spread onto a LB agar plate containing appropriate antibiotic(s) or substrate(s). Colonies usually appear after 12-16 hr of incubation at 37 °C. 58 2.3.8. Shuttling of broad-range plasmids between E. coli and A. tumefaciens or R. meliloti Introduction of broad-range plasmids into Agrobacterium tumefaciens or Rhizobium meliloti were carried out by triparental mating (Ditta et al., 1980) or electroporation (Cangelosi et al., 1991). Triparental mating was conducted by mixing equal proportions of helper strain MT616, donor strain and recipient strain cells together on MG/L (for Agrobacterium tumefaciens) or LB/MC (for Rhizobium meliloti) agar plate and incubating the plate overnight at 28°C. A small amount of the mating mixture was picked and streaked onto an AB or M9/sucrose agar plate containing appropriate antibiotics. The A. tumefaciens exconjugates usually appeared after 2-3 days of incubation while E. coli could not grow on AB or M9/sucrose plates. For triparental mating involving the transfer of plasimd from Agrobacterium into E. coli, a similar procedure was performed. The mating mixture of three parent strains was incubated on an LB plate overnight and then streaked onto an LB plate containing the appropriate antibiotics to select for E.coli containing the plasmid. The plate was cultured overnight at 37 °C since neither A. tumefaciens nor Rhizobium could grow at 37 °C. Electroporation was also used in this study to introduce a plasmid into Agrobacterium. Electrocompetent Agrobacterium cells were prepared as follows. Cells cultured overnight at 28 °C were scraped from the plate with a sterile wooden stick and then transferred into a sterile eppendorf tube. The cells were washed once with ice-cold water and once with ice-cold 15% glycerol. The cell pellet was resuspended in 50-100 µl of ice-cold 15% glycerol and then plasmid DNA (50-100 ng in 10 µl or less of water) was added. The mixture of cells and DNA was transferred 59 to a chilled BioRad electroporation cuvette and kept on ice for 10 min. Gene Pulser II Electroporation System (BioRad) was set to the 25-µF capacitor, voltage of 2.5 kV and controller unit of 400 ohms. The outside of the cuvette was wiped with tissue paper to get rid of moisture before the cuvette was slide into the shocking chamber base. The cells were usually pulsed for 8-10 milli-seconds. Then, ml of MG/L medium was added and the mixture was transferred to an 15-ml culture tube. After culturing at 28 °C for 1h, the cells were collected and spread onto an MG/L plate containing the selectable antibiotics. Colonies usually appeared three days later. 2.4. RNA manipulations 2.4.1. RNA isolation from mammalian cells Total RNA of mammalian cells was prepared using TRIZOL Reagent (GIBCO/Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. In brief, mammalian cells from one 75 cm2 flask were washed once with 10 ml of PBS before ml of TRIZOL Reagent were added. The homogenized sample was then vortexed for 30 s and incubated at room temperature for min. Residual protein was removed after the steps of addition of 400 µl of chloroform, mixing for 30 s, incubation at room temperature for min, and centrifugation for 15 at 12000 × g and °C. The RNA in the colorless aqueous phase was precipitated in ml of isopropanol by mixing for 15 s, incubation for 10 at room temperature, and centrifugation for 10 at 12000×g and °C. The resulting RNA pellet was washed with ml of 75% ethanol and centrifuged for at 7500 × g and °C. The RNA pellet was air dried, resuspended in DEPC-treated water, and stored at -80 °C. The extracted RNA was treated with DNase before the RT-PCR was conducted. 60 2.4.2. RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) was carried out using QIAGEN onestep RT-PCR kit (QIAGEN). The 25 µl RT-PCR reaction mixture was comprised of the following components: × RT-PCR buffer µl RNase-free water 11 µl Primer (10 pmol/µl) 0.5 µl Primer (10 pmol/µl) 0.5 µl dNTP Mix µl RNase inhibitor µl RNA template µl QIAGEN Onestep RT-PCR Enzyme Mix µl 61 The RT-PCR were run using the following program: cycle 50 °C for 30 cycle 95 °C for 15 40 cycles 94 °C for Annealing at (Tm-5) °C for 30 seconds Extension at 72 °C for per kb cycle 72 °C for 10 2.5. Southern blot analysis Southern blot analysis was carried out using the ECL random prime labeling and detection system (Amersham life science) following the instructions of the manufacturer. 2.5.1. Labeling of probes with fluorescein Probes were labeled randomly with fluorescein (Amersham) as described below. Plasmid DNA was digested with the appropriate restriction enzyme(s). The target DNA fragment was isolated from the agarose gel after electrophesis and purified by QIAquick Gel Extraction Kit (QIAGEN). The DNA fragment (50-500 ng in 34 µl of water) was denatured by boiling for and chilled on ice. Then the nucleotide mix (10µl), random primers (5 µl) and Klenow enzyme solution (1 µl) were added and mixed by gently pipetting. The reaction mix was incubated at 37 °C for one hour 62 and terminated by the addition of EDTA to a final concentration of 20 mM. The probes could be stored at – 30 °C in the dark for one year till needed. 2.5.2. Membrane blot preparation Genomic DNA of A. tumefaciens or Rhizobium meliloti was digested and then separated in an 1.0% agarose gel. The agarose gel was depurinated by incubation in the depurination solution (0.25 M HCl) at room temperature for 10 with agitation and followed by denaturation (denaturation solution: 1.5 M NaCl, 0.5 M NaOH) for 25 and neutralization (neutralization solution: 1.5 M NaCl, 0.5M Tris-HCl, pH 7.5) for 30 min. The PosiBlot 30-30 Pressure Blotter (Stratagene) was assembled with the sponge (pre-wet by 10 × SSC), filter paper, mask with window, agarose gel and nylon membrane inside from up to bottom. The DNA was transferred onto the nylon membrane at a constant pressure of 60-75 Hg for 1-2 h by using 10 × SSC (0.15 M Na3Citrate, 1.5 M NaCl) as the transfer buffer. Thereafter, the DNA fragments were fixed on the membrane by UV crosslinking for min. 2.5.3. Hybridization and stringent wash The blots were put into 15 ml of hybridization buffer (5 × SSC, 0.1% SDS, 5% dextran sulphate, 20 × dilution of liquid block) preheated to 65 °C and prehybridized for h with rolling in a hybridization oven (HYBAID). Then, denatured probe were added and hybridization was conducted at 65 °C for overnight as above. The membrane was transferred into an excess volume of washing solution (1×SSC, 0.1% SDS) and washed for 15 twice at 65 °C with gentle agitation. One further wash was carried out using 0.5 × SSC, 0.1% SDS as the washing solution. 63 2.5.4. Blocking, antibody incubation and washing The above treated blot was then transferred to a clean container and rinsed with buffer A (100 mM Tris-HCl, 600 mM NaCl, pH 7.5) for min. Blocking was conducted by incubating the membrane in an 20-fold diluted of liquid block in buffer A (100 mM Tris-HCl, 600 mM NaCl, pH 7.5) for 30 at room temperature. The blot was then put in an appropriately sized container and incubated in diluted conjugate solution (1000-fold diluted anti-fluorescein-HRP conjugate in freshly prepared 0.5% BSA in buffer A) for 30 min. The unbound conjugate was removed by three washes in excess of 0.1% Tween-20 in buffer A for 10 each. 2.5.5. Signal generation and detection The washed blot was placed in premixed detection solution I and II at equal volume and incubated for min. The blot was then transferred to a film cassette and a sheet of autoradiograpy film was exposed for an appropriate length of time before the film was developed in a KODAK developing machine. 2.6. Protein techniques 2.6.1. Buffers for protein manipulations Buffers used in protein manipulations are listed in Table 2.5. 2.6.2. SDS-PAGE gel electrophoresis Protein profiles were analyzed using SDS-PAGE (Laemmli, 1970) based on molecular weight. The electrophoresis apparatus used was the Mini-Protean III Electrophoresis Cell (BioRad). The apparatus was assembled according to the instructions provided by the manufacturer. The monomer stock solution of 64 Table 2.5. Buffers used in protein manipulations Name Components (for L) pH adjustment 10 × Tris-buffered saline 0.2 M Tris base Adjust pH to 7.6 (10 × TBS) 1.37 M Sodium chloride 38 ml 1M Hydrochloric acid × TBST 0.1% Tween-20 (v/v) in × TBS 10 × Tank buffer 0.25 M Tris 1.92 M Glycine No need to check pH 0.1% SDS 10 × Transfer buffer 48 mM Tris Adjust pH to 8.3 38 mM Glycine 0.37 g SDS 20% Methanol × Separating gel buffer 1.5 M Tris-HCl Adjust pH to 8.8 × Stacking gel buffer 0.5 M Tris-HCl Adjust pH to 6.8 × SDS gel-loading buffer 50 mM Tris-HCl (pH 6.8) 100 mM Dithiothreitol 2% SDS 0.1% Bromophenol blue 20% Glycerol Staining solution 0.25 g Coomassie Brilliant blue R (Gibco) 400 ml Methanol 70 ml Acetic acid Destaining solution I 400 ml Methanol 70 ml Acetic acid Destaining solution II 70 ml Acetic acid 50 ml Methanol 65 acrylamide/bis-acrylamide (30.8%T/2.7%C) was prepared as described in Molecular Cloning (Sambrook et al., 1989) and stored in dark at °C. Ammonium Persulfate (APS) (10%) solution was freshly prepared before each use. Separating gel buffer (4 ×, 1.5 M Tris-HCl, pH 8.8) and stacking gel buffer (4 ×, 0.5 M Tris-HCl, pH 6.8) were stored at room temperature. Tank buffer was prepared as a 10 × stock solution (0.25 M Tris-HCl, 1.92 M glycine, 1% SDS, pH8.3) and stored at room temperature. Gel loading buffer (2 ×, 100 mM Tris-HCl, pH6.8, 4% SDS, 0.2% bromophenol blue and 20% glycerol, 0.2 M DTT) was prepared without DTT and stored at room temperature. DTT was added from a M stock solution and stored at -20°C before use. The preparation of polyacrylamide gel and the separation of proteins were performed following the instructions of Hoefer Scientific Instruments (Protein electrophoresis-applications guide, 1994). In this study, 12% PAGE gel was used for the analysis of proteins unless otherwise specified. 2.6.3. Staining of SDS-PAGE separated proteins with standard Coomassie blue SDS-PAGE separated proteins were stained according to the instructions of Hoefer Scientific Instruments (Protein electrophoresis-applications guide, 1994). The gel was placed in the staining solution and shaked at low speed for h. The staining solution was then discarded and replaced with destaining solution I. After the gel had been destained for 30 min, destaining solution I was removed and replaced with destaining solution II. The destaining solution II was changed twice a day until the gel background was clear. 2.6.4. Western blot analysis The sample was mixed with equal volume of × loading dye buffer (Laemmli, 1970), and boiled in a water bath for 5-10 min. After cooling down, the sample was 66 loaded into a SDS/12% polyacrylamide gel, and separated at a constant voltage of 100V. The protein was transferred to an Immun-BlotTM PVDF membranes (Bio-Rad) from the gel in mini gel transfer system for hr to overnight at 200 mA before the non-specific binding sites were blocked by immersing the membrane in 10 % non-fat milk (Nestle) in TBST for h at room temperature on an orbital shaker. The membrane was then washed in TBST buffer for 3×10 min, and incubated in the diluted primary antibody for h at room temperature. The membrane was washed three times as above before incubation in the diluted secondary antibody for h at room temperature. After washing thoroughly as above, the membrane was processed for signal detection according to the recommendations of the manufacturer (Amersham). 2.7. Transposon tagging A. tumefaciens A348 and E. coli S17.1(λpir) with target transposon were freshly grown as the recipient and donor respectively. The cell density was × 10 cells per ml for both the donor and recipient, but the ratio of the cell suspension volume for the donor to the recipient was 1:4. After the mixture was spotted on the LB plates, the plates were dried for h at 37°C and then incubated at 28°C for 24 h. Exconjugants were selected on minimal medium AB plates containing 100 µg ml-1 of gentamycin. The plates were incubated at 28°C for days to select for the transposon mutants, since gentamycin resistance is encoded by the transposon that can replicate in E. coli S17.1(λpir) but not in A. tumefaciens that is sensitive to gentamycin. 67 2.8. General protocol for gene transfer into mammalian cells 2.8.1. Preparation of bacteria E. coli, A. tumefaciens and R. meliloti cells were grown overnight in LB ,MG/L or LBMC liquid medium supplemented with the appropriate antibiotics before they were harvested from the liquid culture by centrifugation at 10,000 g. The bacterial cells were washed with PBS and diluted to an OD600 of 0.2-0.3 for induction or gene transfer assay. For A. tumefaciens vir genes induction, cells were resuspended to an OD600 of 0.2 to 0.3 in IB (Cangelosi et al., 1991) liquid medium in the presence of 100 µM acetosyringone (AS). The cell cultures were incubated at 28 °C for 18-20 h with shaking. 2.8.2. Preparation of mammalian cells Mammalian cells were trypsinized and resupended in fresh DMEM with antibiotics and then seeded at ×106 cells in each well of an 6-well tissue culture plate. The cells were then incubated at 37°C in an 5% CO2 incubator for 18 h. About h before incubation, the cells were washed once with PBS and incubated in fresh medium without antibiotics. For the study of the staining pattern of rhodamine phalloidin, mammalian cells were seeded on the glass cover-slip in 6-well tissue culture plates. 2.8.3. Agrobacterium-mediated gene transfer into mammalian cell Mammalian cells were cocultured with A. tumefaciens by adding 200 µl of bacterium cells (about 4x107 bacteria) to each of the well tissue-culture plates 68 containing the mammalian cells and incubated at room temperature for h. Then the plates were transferred to an 37°C incubator with 5% CO2 . After an overnight incubation, the mammalian cells were washed with PBS for three times to remove the no-specifically bound bacterial cells before they were incubated in ml of complete medium supplemented with mM cefotaxime, which would kill bacterial cells. These cells were used subsequently for later assays. 2.8.4. Attachment and internalization assays To measure the number of bacteria adhering to the mammalian cells, the 6-well plates were washed times with PBS solution before the mammalian cells were lysed with 1% (v/v) Triton X-100 in PBS. The number of bacteria was quantified by agar plate culturing and counting. To measure the internalization of bacteria, the mammalian cells were washed three times with PBS and then incubated for an additional h in tissue culture medium containing 100µg/ml gentamycin (Sigma) to kill extracellular bacteria. After incubation, the mammalian cells were washed twice with PBS solution and lysed with 1% Triton X-100 in PBS before the numbers of bacteria were quantified as above. The adhesion and invasion rates were calculated from the average of three independent experiments. 2.8.5. Observation of gene transfer events 2.8.5.1. Fluorescence microscope analysis To detect the transient expression of egfp, the mammalian cells were examined with a Zeiss Axioskop microscope equipped with a filter set for GFP (exciter filter BP 480nm, banner filter 510-520nm) using 40 X plan neuflor objectives 36 h after incubation. Photographs were recorded on Fuji 100 colour film. 69 2.8.5.2. Flow cytometry analysis EcoPack2-293 Packaging Cells were resuspended in PBS and filtered through 10 µm of nylon before examined under a FACS cytometer, using 100 mW at 488 nm. The fluorescence intensity from 100,000 cells was recorded. 2.8.6. Confocal microscopy analysis Mammalian cells were seeded on the glass coverslips in 6-well tissue culture plates and incubated with the Agrobacterium harbouring pQM49, which express gfpuv constitutively. After h incubation, the mammalian cells were washed with PBS and examined under a confocal microscope. A blue laser of excitation wavelength 488 nm was used to view Agrobacterium cells expressing GFPuv. Optical sectioning of 293 cells was carried out serially at 0.8 µm intervals along the apical and basal axies of the cells. 2.8.7. Observation of actins in mammalian cells Rhodamine phalloidin (Molecular Probes) was used to label actin of mammalian cells according to the manufacturer’s instructions. In brief, mammalian cells were fixed for 10 with PBS/3.7% paraformaldehyde at room temperature (RT) and then washed twice with cold PBS/0.1% BSA. After permeabilization for 10 with PBS/0.1%BSA/0.1%Triton X-100 at RT, the cells were equilibrated by incubation with PBS/2%BSA for 10 at RT and then incubated with phalloidinrhodamine (Molecular Probes, Eugene, OR) (5 µl /200 µl PBS/1%BSA) for h at RT in the dark. After staining, the coverslips were washed by PBS for times. Labelled actin was visualized under a confocal laser scanning microscope using an excitation wavelength of 514 nm. 70 2.8.8. Internalization and gene transfer inhibition assay The cytoskeleton inhibitors, Cytochalasin B and D, were used to study the mechanism of Agrobacterium internalization and gene transfer. Dimethyl sulfoxide (DMSO) was used to dissolve all chemicals. Prior to use, the inhibitors were further diluted in DMEM (2 µg/ml) and added to the mammalian cell culture. After h of incubation, the cells were used for gene transfer as described. 2.9. General transformation and internalization of plant cells Agrobacterium cells were grown in MG/L liquid medium for overnight. Then they were resuspended to an OD600 of 0.2 to 0.3 in IB liquid medium in the presence of 100 µM AS for 16 h. After induction, the bacterial cells were washed with MS medium twice. BY2 used for transformation or invasion assay were subcultured days before. Finally, two hundred µl of the prepared bacterial suspension (OD600=0.2) was mixed with ml of BY-2 cells (4×105) in a petri dish. The internalization assay was carried out as described as that of mammalian cells. For GUS Assays, the cocultivation mixture were pelleted and incubated in the staining solution containing 100 mM sodium phosphate buffer (pH 7), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM Na2EDTA, 0.5 % (v/v) Triton X-100 and 5-bromo-4chloro-3-indolyl β-D-glucuronide (X-Gluc) at 0.5 mg/l for to hours at 37°C to obtain the final staining results 2.10. Statistical analysis All data from attachment, internalization and gene transfer assays were expressed as mean ± standard deviation (SD). The data were analyzed using excel (Miscrosoft Software, USA). 71 [...]... genes induction, cells were resuspended to an OD600 of 0 .2 to 0.3 in IB (Cangelosi et al., 1991) liquid medium in the presence of 100 µM acetosyringone (AS) The cell cultures were incubated at 28 °C for 18 -20 h with shaking 2. 8 .2 Preparation of mammalian cells Mammalian cells were trypsinized and resupended in fresh DMEM with antibiotics and then seeded at 1 ×106 cells in each well of an 6-well tissue... plate The cells were then incubated at 37°C in an 5% CO2 incubator for 18 h About 4 h before incubation, the cells were washed once with PBS and incubated in fresh medium without antibiotics For the study of the staining pattern of rhodamine phalloidin, mammalian cells were seeded on the glass cover-slip in 6-well tissue culture plates 2. 8.3 Agrobacterium- mediated gene transfer into mammalian cell Mammalian. .. 10 µl or less of water) was added The mixture of cells and DNA was transferred 59 to a chilled BioRad electroporation cuvette and kept on ice for 10 min Gene Pulser II Electroporation System (BioRad) was set to the 25 -µF capacitor, voltage of 2. 5 kV and controller unit of 400 ohms The outside of the cuvette was wiped with tissue paper to get rid of moisture before the cuvette was slide into the shocking... The cells were usually pulsed for 8-10 milli-seconds Then, 1 ml of MG/L medium was added and the mixture was transferred to an 15-ml culture tube After culturing at 28 °C for 1h, the cells were collected and spread onto an MG/L plate containing the selectable antibiotics Colonies usually appeared three days later 2. 4 RNA manipulations 2. 4.1 RNA isolation from mammalian cells Total RNA of mammalian cells. .. sectioning of 29 3 cells was carried out serially at 0.8 µm intervals along the apical and basal axies of the cells 2. 8.7 Observation of actins in mammalian cells Rhodamine phalloidin (Molecular Probes) was used to label actin of mammalian cells according to the manufacturer’s instructions In brief, mammalian cells were fixed for 10 min with PBS/3.7% paraformaldehyde at room temperature (RT) and then washed... Internalization and gene transfer inhibition assay The cytoskeleton inhibitors, Cytochalasin B and D, were used to study the mechanism of Agrobacterium internalization and gene transfer Dimethyl sulfoxide (DMSO) was used to dissolve all chemicals Prior to use, the inhibitors were further diluted in DMEM (2 µg/ml) and added to the mammalian cell culture After 4 h of incubation, the cells were used for gene transfer. .. transfer into mammalian cell Mammalian cells were cocultured with A tumefaciens by adding 20 0 µl of bacterium cells (about 4x107 bacteria) to each of the 6 well tissue-culture plates 68 containing the mammalian cells and incubated at room temperature for 4 h Then the plates were transferred to an 37°C incubator with 5% CO2 After an overnight incubation, the mammalian cells were washed with PBS for three... In brief, mammalian cells from one 75 cm2 flask were washed once with 10 ml of PBS before 2 ml of TRIZOL Reagent were added The homogenized sample was then vortexed for 30 s and incubated at room temperature for 5 min Residual protein was removed after the steps of addition of 400 µl of chloroform, mixing for 30 s, incubation at room temperature for 3 min, and centrifugation for 15 min at 120 00 × g... incubated at 28 °C for 24 h Exconjugants were selected on minimal medium AB plates containing 100 µg ml-1 of gentamycin The plates were incubated at 28 °C for 2 days to select for the transposon mutants, since gentamycin resistance is encoded by the transposon that can replicate in E coli S17.1(λpir) but not in A tumefaciens that is sensitive to gentamycin 67 2. 8 General protocol for gene transfer into mammalian. .. cells were used for gene transfer as described 2. 9 General transformation and internalization of plant cells Agrobacterium cells were grown in MG/L liquid medium for overnight Then they were resuspended to an OD600 of 0 .2 to 0.3 in IB liquid medium in the presence of 100 µM AS for 16 h After induction, the bacterial cells were washed with MS medium twice BY2 used for transformation or invasion assay were . dH 2 O, filter sterilized 24 24 24 X-Gal Dissolved in dimethyl sulfoxide 20 20 20 Proteinase K Dissolved in dH 2 O 20 50 50 RNase Dissolved in dH 2 O 10 20 20 51 Table 2. 4 1991 20 × AB salts NH 4 Cl, 20 g; MgSO 4 . 7H 2 O, 6 g; KCl, 3 g; CaCl 2 , 0 .2 g; Fe SO 4 . 7H 2 O, 50 mg. Cangelosi et al., 1991 20 × AB buffer K 2 HPO 4 , 60 g; NaH 2 PO 4 , 23 g;. PIPS, 55 mM MnCl 2 , 15 mM CaCl 2 , 25 0mM KCl; MG/L LB, 500 ml; mannitol, 10 g; g sodium glutamate, 2. 32; KH 2 PO 4 , 0.5 g; NaCl, 0 .2 g; MgSO 4 . 7H 2 O, 0 .2 g; biotin, 2 µg; pH 7.0. Cangelosi