Transfection of Sf9/Sf21 Cells

[Garry Myers]

Hi,

I've recently become the proud owner of the CLONTECH BacPAK expression
system ;). The protocol accompanying the kit suggests using Lipofectin(tm)
to transfect the Sf21 cells with recombinant virus. I'm interested to hear
any thoughts or opinions from baculoviral-minded individuals on other
methods of transfecting Sf9/Sf21 cells, particularly electroporation.

Cheers,

Garry.

___________________________________________________________________
Garry Myers Menzies School of Health Research
Molecular Genetics Unit ga...@menzies.su.edu.au
___________________________________________________________________

[po...@molbiol.ox.ac.uk]

[po...@molbiol.ox.ac.uk]

In article <garry.23...@menzies.su.edu.au>, ga...@menzies.su.edu.au (Garry Myers) writes:

> I've recently become the proud owner of the CLONTECH BacPAK expression
> system ;). The protocol accompanying the kit suggests using Lipofectin(tm)
> to transfect the Sf21 cells with recombinant virus. I'm interested to hear
> any thoughts or opinions from baculoviral-minded individuals on other
> methods of transfecting Sf9/Sf21 cells, particularly electroporation.

> ___________________________________________________________________
> Garry Myers Menzies School of Health Research
> Molecular Genetics Unit ga...@menzies.su.edu.au
> ___________________________________________________________________

Hi there,

Sorry for the other abortive post.

Electroporation works fine for Sf21 cells but efficiency depends on several
parameters. However, production of recombinant baculoviruses by cotransfection
of transfer vectors and linearised 'wild type' baculovirus DNA into Sf cells
is very efficient and much simpler using lipofectin ( 5 microliters for 35 mm
dishes is enough).

If you wish to try electroporation: we've used-

using a BRL electroporator (trademark etc) at 1600 uF
with 0.4 cm electrode seperation and 1 ml cuvettes

resuspend cells in chilled HEPES buffered glucose saline
and pulse to give 60-80% cell viability by trypan exclusion.
DNA uptake is maximal at 65-70% and is quite poor outside
60-80%. Also keep cell conc. < 3 million per ml as excess cell fusion
results/ and low DNA uptake.

550 volts/cm fieldstrength with the above conditions worked for us

in our hands electroporation was 100 times better than
calcium phosphate and 3-5 times better than lipofectin
for introducing viral DNA into Sf21 cells

I can supply more details if required.

Best of luck

Ian

Ian Polkinghorne PO...@molbiol.ox.ac.uk NOTE: All comments are mine
NERC Institute of Virology and do not represent NERC,
& Environmental Microbiology Oxford Uni, IVEM, or the
Mansfield Rd Oxford OX1 3SR UK QLD DPI.. etc and so forth
(Just another Aussie)

[Hinayana Bawagan]

ga...@menzies.su.edu.au (Garry Myers) writes:

>Hi,

>Cheers,

>Garry.

This article may be useful to you.

Journal of General Virology (1989, 70, 3501-3505.

Efficient transfection of insect cells with baculovirus DNA using
electroporation. By Susan G. Mann and Linda A. King.

[Elmer M. Price, Ph.D.]

In article <garry.23...@menzies.su.edu.au>

Hi Garry-I would strongly recommend against electroporation as a means
of transfecting insect cells. The point of the transfection is to
introduce two DNA molecules into the insect cell-one being the transfer
vector and the other being the 140Kb baculoviral DNA. Once inside the cell,
the miracle of homologous recombination occurs and recombinant baculovirus
is generated. This is not too terribly efficient and in order to optimize
the entire process, one needs to optimize the co-transfection process. The
lipofection process works very well, while electroporation is probably (IMHO)
not as efficient at co-transfection (but great for single transfections).
The lipofection step is also very easy, taking only about 30 minutes of effort
on the part of the investigator.

Hope this helps

Elmer Price, Ph.D.