실험 9. In vitro transcription and transfection of Sindbis RNA genomes

[colony]

I. Introduction

1. Alphavirus - Togaviridae family -Sindbis virus, Semliki forest Virus

2. Expression‎ of cloned sequences using viral vector

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Insect cell- restricted baculovirus vector
Vaccinia virus vector
Episomal plasmid vector based on the Epstein-Barr virus
Retroviral vector
Alphavirus based vector
 

3. Sindbis virus biology

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One of the least pathagenic of the alphaviruses
Host range - SIN can infect a variety of cells including those of insect avian, mammalian, amphibian and reptilian origin. In practice, however, there are limitations host range. The basis for its selectivity is not completely understood and may involve several steps of the viral life cycle
 

4. SIN genomic RNA

5. Transfection of DNA into eukaryotic cells

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Calcium phosphate transfection
Transfection using DEAE-Dextran
Electroporation and Lipofection -this Experiment

II. Materials

1. Recombinant Sindbis viral genome (ToTo-EGFP, rdSIN-EGFP) and Helper DNA (SIN-ST)
2. Baby Hamster Kidney cell(BHK-21)
3. PBS(phosphate buffered saline) (per liter)
         NaCl               10.00g
         KCl                   0.24g
         Na₂HPO₄          1.44g
         KH₂PO₄            0.25g  pH 7.0
         bottle in 400ml amounts. Autoclave to sterilize
4. DMEM - fetal calf serum(10%)
5. Tissue culture flasks and dishes
6. Lipofectin
7. 10x Transcriptional buffer
8. 10mM rNTP
9. 2.5uM CAP analogue
10. Sp6 RNA polymerase
11. RNasin
12. DEPC treated water

III. Experimental Procedures

1. In vitro transcription

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The reaction can be scaled up or down depending on how much RNA is needed. Typical yield is 1-5 ug RNA per ug of DNA for a 13 kb plasmid. Extreme care should be taken to avoid cantaination of reagent.

Linearize DNA with appropriate restriction enzyme(if possible). Clean up DNA using RNAse free technique and precipitate. Do not add tRNA as a carrier.
Transcripted RNA reaction mixture.
       DNA                                           2ug (6ul)   
       10x Transcriptional buffer         2.5ul
       10mM rNTP                                2.5ul
       2.5mM CAP analogue                   5ul
       Sp6 RNA polymerase                 0.7ul
       RNasin                                       0.7ul
       DEPC treated water                   7.6ul
    -----------------------------------------------------
          Total                                         25ul

Incubate the mixture at 38-40℃ water bath for 1 hr.
Optimal : Phenol/chloroform extract, precipitate, resuspend for transfection. (DNA can be dagraded using RNAse - free DNAse I, but yield will decrease. The plasmid DNA is not infectious, and there is usually no need to get rid of it.)
 

2. Transfection using lipofectin

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Mix transcripted RNA mixture 25 ul with PBS 275 ul.
: Usually, the half amount of transcripted RNA at 25 ul reaction is used for
  one of the six well plate.
Mix PBS 270 ul with lipofectin 30 ul.
Add diluted RNA mixture to diluted lipofectin tube Just by tapping.
Incubate the mixture on ice for 10 min before transfection.
Remove the DMEM medium of semi - confluent BHK-21 monolayers(6-well plate) and wash the plates with 2 ml PBS.
Add 150 ul of the RNA - lipofectin mix with dropwise in each well. Incubate the plates, at RT, rocking occasionally for 10 min.
Remove the RNA - lipofectin mix, wash the monolayers 2 times with 1 ml PBS.
Add 2 ml of DMEM + 10% FCS medium to plates.
Incubate the plates at 37℃ for 36 - 40 hr.
Observe the state of cells using microscope.
Harvest the cell using the scraper.
Centrifuge and then store the supernatant.
The virus yields will be checked by plaque assay.
The Sindbis expression‎ system can be used to express recombinant protein in a variety of cell lines. BHK cells, however, have been shown to yield the maximum levels of protein. To study the effects of your gene in another cell line, recommend that a) optimize transfection to maximize the number of cells expressing your recombinant RNA. b) perform a time course of expression‎ as host cell functions after the Sindbis life cycles.

IV. Notes

1. Transfection using electroporation

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Grow BHK cells in a 175 cm² tissue culture flask containg 30 ml complete DMEM.
Aspirate medium and wash cells once with PBS.
Add 3 ml of trysin/EDTA solution and incubate for 1-2 min at 37℃.
Add 10 ml of complete DMEM to stop trypsinization and briefly pipet the solution to obtain a single cell suspension.
Centrifuge the cells at 400x g for 5 min at RT and aspirate supematant. Resuspend cells in 10 ml of DMEM.
Centrifuge the cells at 400x g for 5 min at RT and aspirate supernatant. Resuspend cells in 10 ml of PBS without cations. Determine the number of cells with a hemocytometer.
Centrifuge the cells at 400x g for 5 min at RT and aspirate supernatant. Resuspend cells in 10 ml of PBS without cations at a concentration of 10cells/ml
Place 0.5 ml(5x10⁶cells) of cell suspension into a 0.4cm electroporation cuvette.
Add 5-10 ul of the transcription reaction(5-20 ug of DNA) to the cell suspension.
set up electroporation device to give a field strength of 1.5kV/cm with 25uF capacitance.
Place the cuvette in the electrophorator and pulse the cell suspension twice. Wait until the unit recharges itself before pulsing again. Place cells on ice for 5 min to allow cells to recover.
Trasfer electroporated cells (0.5 ml) to 9.5 ml complete DMEM medium. rinse the cuvette with the suspension to collect all the cells.
Plate the cells (1.5 ml on a 35mm plate, 3 ml on a 60 mm plate, 7 ml on a 100 mm plate).
Wash cells warmed media(1 ml) at 12hr post-transfection.
Harvest media at 30-48 hr post-transfection. store at 4℃.
The virus yields will be checked by Green fluorescence.

V. References

1. Bernard N. Fields and David M. knipe (1990) Virology Chapter 25.
2. Ausubel F. M., et al (1994) Current protocols in Molecular biology unit 9.3-9.4
3. Hahn, Y.S. et al (1992) Proc. Natl. Acad. Sci. USA 89 2679-2683
4. Schlesinger, S (1993) Trends Biotechnol. 11 18-22
5. Xiong, et al (1989) Science 243 1188-1191

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