Calcium Phosphate Precipitation

brett brett at BORCIM.WUSTL.EDU
Mon Feb 17 12:55:18 EST 1997


>mennow at ruly46.medfac.leidenuniv.nl wrote:
>> 
>> Does anyone have experience with calcium phosphate precipitating DNA to
>>macrophages (J774).
>> I have been trying to get a GFP vector in the cells last weeks, for
>>transient expression. But I never got any signal.
>> While the vector works perfectly in ES-cells. Is it possible that the
>>macrophages just eat the calcium phosphate particles with the DNA?
>> I want to use the GFP as a marker to check transfection together with a
>>macrophage specific construct.
>> Does anyone have any suggestions on this, or other ways to test a macrophage
>>specific construct in vitro.
>> 
>> Menno de Winther
>Hi Menno,
>
>I've never used macrophages for transient expression as we use HEK 293's
>as a rule. But I foulnd that the same vector can give very different
>expression levels in different cell lines. We get transfection
>efficiencies of up to 40% with HEKs but only 1-2% with CHO, for
>instance. So it may simply be that your macrophages don't like the
>promoter on that vector. Have other vectors been used on your cell line?
>
>HOpe it helps
>Andy D
>-- 

I got G418-resistant P388.D1 and RAW264.7 mac lines using BES-buffered
calcium phosphate transfection, ala Red Book, using a bicistronic vector
based on the CMV promoter. Expression of my protein was low, however. Also,
I tried J774s, but they were never transduced to neoR.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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