Pete,
I work with the Baculovirus system so all of my comments are based on this
system.
The short answer to your questions is that they are all system dependent.
>1.) At what frequency do recombinants appear?
>>2.) Do you think that cell type will drastically effect recombinant
>frequency? (i.e.: can I assume that data from one system is in the same
>ballpark?).
The frequency of recombination will vary depending on: 1) the method used for
transfection, 2) the cells being transfected, and 3) the condition of the
plasmid DNA being used in the transfection. However, the first two variables
will have the greatest impact on your transfection results. Each cell line has
a different susceptibility for transfection. For example in the baculovirus
system, the cell line SF21 AE is between 10-100 fold more transfectable (if
that's a word?) than the cell line TN368.
>3.) Does any particular transfection method (electroporation,
>liposome-mediated, CaPhosphate, or others) work better than others?
I have only worked with Calcium Phosphate precipitation in my transfections.
The yields of recombinant virus using this method were fairly low (<5%).
However, several coworkers have tried both electroporation and lipofection and
gotten much better results. In some cases, the yields have been greater than
30%. I would therefore recommend one of these methods over Calcium Phosphate
precipitation. I know that electroporation requires buying an electroporator.
I don't think there is any equipment purchases for lipofection.
>4.) How much homologous DNA seems to be necessary to achieve an
>observable recombinant frequency?
Amounts of DNA are really minimal. I routinely used between 0.1-1 ug of DNA /
reaction.
There are two good reference books for the baculovirus expression system which
go into great detail about transfection procedures and selection of recombinant
viruses. One was written by Lois Miller and the other by Max Summers. In
addition, if you are at the Madison campus then I would recommend that you
contact Dr. Paul Friesen. He worked for Lois Miller and has been doing
transfections for a number of years.
Good Luck
Bart Corsaro
Lederle-Praxis Biologicals
West Henrietta, NY
EMail: Bart_Corsaro_at_USLRMG01 at internetmail.pr.cyanamid.com
