transfecting siRNA into primary fetal mouse hepatocytes - (Apr/16/2008 )

I am trying to knockdown some genes in primary fetal mouse hepatocytes isolated from E16 embryos, strain: C57BL6

The method of isolation is by collagenase digestion followed by ACK buffer to lyse the RBC.

To evaluate transfection efficiency, I use an siRNA against GAPDH labelled with FAM and observe under fluorescent mic

The first reagent I used is siPORT Lipid (Ambion), I transfected them the day after isolation and overnight attachment of 2 x 10(5) cells per well in 12-well collagen coated plate (BD Biocoat).

No fluorescent positive cells, and there also seem not to be any toxicity.

So I then switched to electroporation, with AMAXA, primary mouse hepatocyte neurotransfection kit

I tried 0, 25, 50, 100, 200nM siRNA, cell number is 1x10(6) cells per reaction and there seem to be a lot of dead cells after electroporation.

The fluorescence are all in the flowing cells after I plate them and they will washed off when I change the medium (4 hrs after electroporation)



Now...should I try siPORT lipid again??

I ran out of ideas.

What you expect to see with FAM-labeled siRNA and the transfection reagent in cells after transfection is called cellular uptake. It should be a lot uptake due to non-specific charge-charge interaction of reagent with the treated cells. However, that does not tell you much about transfection efficiency, because only a small fraction of siRNA taken up by the cells will be released into cytoplasm and become biologically active. most end up in degradation factory lysosome and inactivated.

Something was not done right. It looks like either you mis-calculated the amount of reagent or siRNA such that you could not even see the uptake. After transfection you should see nearly 100% cells labeled with FAM-siRNA if you used the right amount of siRNA and correct reagent to siRNA ratios.

Thanks for your comments.

The main problem is I dunno how does siPORT lipid works with primary cells.

If I understand it right, siPORT lipid depends on cell proliferation for uptake (correct me if I am wrong)

So there are some possibilities,

1. The Opti-Mem I used for transfection doesn't work with my cells during that 4 hrs of incubation?
2. I did the transfection too quickly after isolation, and maybe I should wait one more day before I do lipid transfection?

Can you tell me how the cells take up the lipid-siRNA complex?? Why it should be 100% uptake???

I tried very hard on PubMed but can't find any paper using primary fetal mouse hepatocytes + siRNA

Thanks, I really appreciate any suggestions as I've been helpless for the past 2 months

I dont know the exact chemical composition of this lipid. However, it is most likely cationic by nature. Cell surface carries anionic charges; therefore the lipid-siRNA should have high affinity to this surface. The binding triggers a cellular uptake event. The most predominant uptake mechanism is believed to be endocytosis.

With right siRNA to lipid ratios, you should see nearly 100% of the treated cell take up these complexes, if these cells are seeded evenly.

However, an effective release of these complexes from endosome/lysosome compartments to cytoplasm is cell type dependent and is typically (much) lower than the uptake rate.

You may want to do some experiments on other established hepatocyte line to get yourself familiar with this technique, or call the tech support for some suggestions. Typically you need to optimize the parameters such as the amount of siRNA, the amount of lipids, the number of cells per well and the incubation time before and after addition to the cells.

See what you think....

I did a new set of transfection yesterday.

On a 12-well plate

in 6 wells I used siPORT Lipid
in other 6 wells, I used Lipofectamine 2000

For each of these 6 wells, I have 6 conditions:

-ve control (reagents added without siRNA)
25nM siRNA (FAM labeled)
50nM siRNA (FAM labeled)
75nM siRNA (FAM labeled)
100nM siRNA (FAM labeled)
1.5ug GFP vector (DNA)

After 5 hrs of transfection, I changed the media and looked in the microscope

For the siPORT Lipid ones, NOTHING happened....

For the Lipofectamine 2000, the GFP transfected ones looks good, the cells were green and healthy....
That means the GFP vector is taken up and expressed already

For the siRNA transfected ones, they don't look the same, the green signals are not like the GFP expressed cells, instead I can see some green "spots" all over the cells like stars in the sky, which I have not seen previously, this observation is however not present in the -ve control, therefore, it must not be autofluoresence

So....any thoughts???

This is what you should see after a successful transfection. The bright dots in cytoplasm are complexes taken up by the cells. The complexes are located either within endosomes or lysosomes. Very few staining are in cytoplasm and nuclei. EGFP protein is located in cytoplasm in a diffused form.

it worked!!!!!!

48hr post-transfection, for the lipofectamine 2000 set of experiment

RNA extracted and real-time PCR performed using TaqMan Assay

siRNA Relative Expression
0 nM 100.00
25 nM 20.40
50 nM 20.54
75 nM 32.68
100 nM 25.17

A roughly 80% knockdown, more than anything I expected!!!!!

Didn't even bother to check what happened to the siPORT Lipid set