Molecular basis of agrobacterium mediated gene transfer into mammalian cells 2

Cell culture and culture medium Mammalian and plant cell lines used in this study are listed in Table 2.4.. Dulbecco’s Modified Eagle’s Medium DMEM DMEM was used to culture mammalian c

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

Table 2.1 Bacterial strains and plasmids

Bacterial strain

or plasmid Relevant characteristic(s) Source or reference

Strains

Escherichia coli

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

S17.1(λpir) λpir lysogen, recA, thi, pro, hsdR-M+,

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

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

pSW172 Broad-host-range IncP plasmid containing Plac

and downstream polylinker sequence, TcR Chen and Winans, 1991 pBSL202 Plasmid harboring mini-Tn5 transposon, GmR,

pCB301 Broad-host-range plasmid derived from pBIN19,

KmR

Xiang et al., 1999

pQM45 pCB301 carrying a 3 kb neoegfp fragment from

pNEOEGFP between T-borders

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

Table 2.2 Media preparation

LB (Luria broth) Tryptone, 10 g; yeast extract, 5 g; NaCl, 10

g; pH 7.5

Sambrook et al.,

1989 SOB Tryptone, 20 g; yeast extract, 5 g; NaCl, 0.5

Murashige and Skoog, 1962

a Preparation for 1 liter, and sterilized by autoclaving; b For solid media, 1.5% agar was added; c no autoclaving is necessary

Table 2.3 Antibiotics and other stock solutions used in this study

Concentration (Con.) (mg/ml)

dH2O, filter sterilized

24 24 24

dimethyl sulfoxide

Table 2.4 Mammalian and plant cell lines cell line Relevant characteristic(s) Source or reference

BY-2 Nicotiana tabacum L cv Bright Yellow

2 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

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 5 %

(v/v) CO2 incubator Cells were maintained in 75 cm2 flasks and subcultured at least once every 5 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 5 % 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 2 ml of

overnight culture were collected by centrifugation at 10, 000 rpm (Eppendorf 5417C)

for 1 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, 5 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 5 min The supernatant was then transferred to a clean

eppendorf tube To precipitate the plasmid DNA, 2 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 5 min The cells were washed once with 4 ml of TES (10 mM Tris-HCl, 25 mM EDTA, 150 mM NaCl, pH 8.0) and resuspended in 4 ml of

TE buffer (10 mM Tris-HCl, 25 mM EDTA, pH 8.0) To lyse the cells, 500 µl of 5 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 2 M and then 2 volumes of ethanol were added The DNA pellet was washed once with 70% ethanol

and vacuum dried Genomic DNA was dissolved in 500 µl of distilled water and stored at 4 °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 1 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 4

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:

Add distilled water to a final volume of 50 µl

The PCR was run using the following program:

30 cycles 95 °C for 30 seconds

Annealing at (Tm-5) °C for 30 seconds Extension at 72 °C for 1 min per kb

2.3.5 DNA gel electrophoresis and purification

DNA fragments were electrophoresized in an 1 × 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 3 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 min to dissolve the gel completely For DNA fragments larger than 4 kb or smaller than 500 bp, 1 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 1 min 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 1 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

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 min before they were collected

by centrifugation at 2600 rpm (Eppendorf 5810R) for 5 min at 4 °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 4 °C) and then incubated on ice for 10 min Cells were collected by

centrifugation as above and resuspended in 5 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 2 min, 900 µl of fresh LB medium were added The cultures were incubated at 37 °C for 30 min 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

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

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, 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 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 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 12000 ×

g and 4 °C The RNA in the colorless aqueous phase was precipitated in 1 ml of

isopropanol by mixing for 15 s, incubation for 10 min at room temperature, and

centrifugation for 10 min at 12000×g and 4 °C The resulting RNA pellet was washed

with 1 ml of 75% ethanol and centrifuged for 5 min at 7500 × g and 4 °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