i normally use PEI (polyethylenimine from polysciences). it is very cheap compared to lipofectamine.i do get at least 90% fluorescent. for 10 cm plate ( cells should be 70-80% confluent), 2 hours before the transfection, i remove the media, wash once with PBS and then replace with media and 10% FBS  but without antibiotics (instead of 10ml, i use 6 ml and the following day after transfection i add 9 ml extra media with serum).  i used optimem for diluting the DNA and PEI. the conc of DNA i use is 10.5 ug and PEI is 52.5 ug. mix them separately with the optimem (250 ul per DNA, 250 ul per PEI) and then combine them and vortex for 30 secs and then incubate at rtp for 20 mins and then transfect ur cells

cheers bro!

Dear Khamal, Many thanks for your reply however may I kindly ask which cell you are working with, mines is Neural Progenitor Cess

Thanks

my lab has tried this with helam cells, sima cells (for studying neuroblastoma) and hela.

let me also add that i use the ratio 2:1:1. that my DNA of interest and the GAG/pol and VSG. Also after 16 -24h post transfection, i transfer the cells to 30 degrees celsius to stabilise the viral production. before, i transduce and cells  with the generated viral media i suck the viral media with a syringe and add 5  ug/ml polybrene, with vigorous shaking and then filter it with 0.45 um filters. 

cheers

Great, Khamal but my friends have suggested not to use Serum with OptiMEM as it blocks viral activity thus lenti viral formation. I recently did with DMEM media with FBS & P/S but was unable to get enough quantity of virus. Now today again I did it with OptiMEM media, without FBS & without P/S. Lets see whats comes. Actually iam doing a reporter plasmid today jut to check out, before again I start with CRISPR plasmid.

• it is true that some do not use serum, however; i find no problem with it. i only exclude antibiotics during transfection. 

Khamal, I am sorry to inform you that when yesterday i did it without serum in OptiMEM, as soon as (after 6hrs of lentiviral complex incubation) i replaced the OptiMEM without Serum to DMEM 10% serum all of my cells within 2 minutes came out of the bottom, and died. I don't understand where I am doing mistake. Please suggest.

Let me know you my detail protocol (which I did yesterday):

Day -1: seeding of 11-12 million of 293 cells into 15cm dish.

Day0: @50-60% confluency I changed the DMEM-10%FBS-10%P/S media with the OptiMEM (reduced serum media) without adding P/S & Serum. Put the cells back in incubator for approx. 30 min then I made the DNA complex as:

12.5ug of Target DNA, 12.5 pacakging (psPAX2), 5ug envelop (VSVG) in 900ul Dist. H2O+ 100ul of 100mM CaCl2 added to  1 ml of 2X HBS (= total 2 ml), and incubated after mixing gently for 15 mints. Somewhat turbid coloration was there. Than added all to the 150mm dish. Incubated for 6 hrs (protocol which I am following says to incubate 5-8 hrs and then change with normal media), and then changed to OptiMEM. But all died as soon as I replaced the OptiMEM with DMEM media.

Please let me add up here that the protocol which I follow says to incubate the complex for 1 min but i Did it yesterday for 15 mints, as other google lot of protocol say the later time point. 

Also my protocol says to do everything in DMEM but again seeing lot of protocol I decided to go for OptiMEM

- although I believe these few changes should not matter.

Please suggest. i shall be highly thankful to you for all your kind assistance.

Best,

S

from ur protocol ur DNA conc is very high and ur cell confluency is also low. the total amt of DNA, i use is 10.5 ug which i distribute among my DNA of interest: gag/pol/:vsg (2:1:1). meanining i use 5.25ug of  my DNA of interest, 2.625 ug gag/pol and 2.625 ug vsg for 10 cm plate. also when i remove the old media and wash once with pbs i do it carefully,  because 293 cells do not strongly adhere to plates. again i replace with 6ml media (NB: not optimem) and serum (10%) . the optimem i use to dilute the DNA and the transfection reagents.Also, i have not use cacl2 b4. 

please do contact me shd u have any more query.i will response to it tommorrow evening. i need to go catch my flight back to UK.

Khamal Many thanks for your kind reply. 

1. How to decide that what is the good ratio for lenti-viral transfection. To be honest I have seen different different ratios. intact even more than the 12.5 ug DNA. but if you see he ratio mines is 2.5:2.5:1(DNA:Packaging:Envelop). Do you know what is the basis of or on what criteria we took ratio (whatever). Thanks

2. Next, I wish to ask if suppose like in your case you took 5.25:2.62:2.62. Taking the same amount can we divide the same for 6 well plate correspondingly. for e.g. 10cm plate area= 78.5 while for 6 well plate= 9.5 approx. So can we take the same amount as of 10cm dish and according to surface area of 6 can we add the required amount. Asking this because i have just seen a recommendation for 10cm, 6 cm, & 6 well plate but was not as per the division of surface area, and I don't know how they divided it.

3. Now, to your points; I too add after OPTIMEM transfection.

Have a safe flight. Looking forward to hear you once you are back in Sheffield.

Best, 

S

HELLO

I have arrived safely.  to your questions

1. i have tried some ratios and they did work okay but i find 2:1:1 to be very optimal. In mine perspective i think using twice as much DNA of interest to the packaging virus means there are as DNA for them to be incorporated but not too saturated for them.

2. so mine ratio is 2:1:1. For different plates i use the following DNA conc and diluent. where mine diluent is optimem.

b. T25: 3.5ug / 100uL diluent

c. T75: 10.5ug / 250uL

d. T175: 25ug / 400uL

e. 6-well: 1ug / 50uL

f. 12-well: 500ng / 25uL

g. 96-well: 100ng / 10uL

Also,  as i have explained already, it is this amount of DNA i share it in the ratio 2:1:1.  i use the PEI conc 5x that of the DNA mixture, so for T75 i will use 52.5 ug of PEI..

i hope this helps. Don't hesitate to contact me shd u need further guidance.

cheers bro

Lipofectamine is surely better than CaPO4. Medium is not important, but serum decreases infection efficiency significantly. For transfection it is also not recommended.

Dear Elena Many thanks for your kind reply but before this during my Ph.D I remember it quite sharply that I used 10% FBS Media, however yesterday when I tried with the OptiMEM-No FBS-No P/S, all the cell died after 24 hrs, altghough after 6 hr later I cahnged the OptiMEM with the DMEM+10FBS+P/S meida. Few cells were still alive in the 150mm dish however no florescent was observed (mCherry was used as control) from them I think plasmid virus didn't form.The major problem is that I don't know what ratio of DNA:psAX2:VSVG should I take as my plasmid of interest is of 14kb, although khamal above has given few ratio I am going to try next one of those for 6 well plate first.

Dear Khamal,

many thanks for being so kind and all yopur help but Khamal one thing I would like to mention is that my both plasmids are of 14kb, so should I take more DNA ratio. Many thanks

You have to use OPTI-MEM only for the complex formation (lipofectamine-DNA complex). But the transfected cells can be in FBS 10% at the moment of transfection, if it is better for your cells. I used this protocols for transfecting my CRISPR plasmids with Lipofectamine 2000.

Claudia Many thanks for your kind reply. May I ask what was your DNAs size as I heard of that as per the size I have to change the DNA:psAX2:VSVG ratio. mines is 14KB.

Below is the protocol which I used, but had no viral formation as I used this for mCherry plasmid as control but was no red colouration in cells.

Day -1: seeding of 11-12 million of 293 cells into 15cm dish.

Day0: @50-60% confluency I changed the DMEM-10%FBS-10%P/S media with the OptiMEM (reduced serum media) without adding P/S & Serum. Put the cells back in incubator for approx. 30 min then I made the DNA complex as:

12.5ug of Target DNA, 12.5 pacakging (psPAX2), 5ug envelop (VSVG) in 900ul Dist. H2O+ 100ul of 100mM CaCl2 added to 1 ml of 2X HBS (= total 2 ml), and incubated after mixing gently for 15 mints. Somewhat turbid coloration was there. Than added all to the 150mm dish. Incubated for 6 hrs (protocol which I am following says to incubate 5-8 hrs and then change with normal media), and then changed to DMEM with FBS10%+P/S.

Dear Sayd,

If your cells die without serum in medium it is better to leave them in serum-medium of course. Though 293T fibroblasts are not easy to kill cells. So, first, if such happens you obviously have at least 2 times more million of cells for the passage before transfection. Second, nobody says transfection/infection is blocked with serum, it is being inhibited. You still can have rate sufficient for your experiment with FCS in transfection medium, but DNA-agent complex surely has to be made in serum-free medium or in steril PBS.

Polyethyleimine commercially available is 30-40 000 in size, but there are many other PEIs of smaller molweight. The rule here is the bigger is PEI molecule the less ratio you need for DNA:PEI complex formation, and contrarywise. The rate of PEI toxicity for ratio is the opposite, the bigger is PEI the less toxic it is. Lipofectamine is toxic enough also, and as it forms a sort of suspension of vesicles in water solutions it needs 2-3 times more than 30-40 thou. PEI amount for  1mkg DNA. 14 kb vector is huge thing, it is better to use PEI for such delivery than lipo-based agents. Large-mol. PEI is not good only for suspension cultures as being heavy it drops down to bottom, for the rest it is good and pretty cheap.

Ratio for DNA-agent compelex depending on vector size should be adjusted the same way: the bigger is vector - the more agent you need for its delivery.

One more ratio in transfection/infection efficiency is the more DNA you push onto cells the more percentage you have. And here you are limited with the agent for delivery concentration, not vector because DNA is non-toxic.

As for myself I prefer to make in vitro experiments with normal small plasmid vectors and have a couple of hundred colonies celection to choose 1-2 clones with stable expression than to fish out among very few colonies from huge retroviral v. infection with relatively low expression and loose it from production nevertheless. For in vivo one doesn't have choice - it has to be Retro- or Lenti.

Good luck with experiments,

E.F.

Dear Syed,

We have been generating lentiviruses for investigators for uh....  many years now. We ONLY use CaPO4 transfection, and I fail to recall an instance when poor transfection was an issue. One caveat is that we use 293FT rather than 293T cells.  These are not as readily transfectable as 293T that you use, but they a) attach better and b) do not acidify the medium as much.  I would suggest to, first, do transfection with a GFP plasmid to make sure that you transfect at least 30% of the cells (and you may want to do the same with some  fancy stuff like Lipofectamine 3000 or PEI just for the peace of mind).

I would also suggest to generate, side-by-side, a control virus with a small, easily detectable insert (e.g.,  GFP).

I would start troubleshooting by trying to make sure that:

1) Your vector backbone is compatible with your packaging plasmids (there are some idiosyncrasies between generations of vectors, so check with your backbone manufacturer)

2) The size is not an issue.  Lentivirus should be able to package a genome of up to 8-10 kb with reasonable efficiency.  However, Titer generally decreases with increased size of the construct.

Small GDP encoding construct should help in making sure that 1) or 2) is not an issue.

3) On occasion, we observed small deletions in the promoter upstream of the 5' LTR as a cloning accident.  As this promoter drives transcription of the genomic RNA for packaging, such a deletion would have a devastating effect on your virus titer.  Sequencing, fine restriction endonuclease mapping, re-making the construct, or even picking another, independent clone for plasmid isolation may help in fixing this problem.

Good luck!

.

Yes, Lipofectamin is more efficient. However I do recomend JetPrime. You can do the transfection with the complete media, in presence of serum and antibiotics and it is much cheaper. The transfection yield is over 90% with 293FT cells.

Dear All,

Long time ago (15 years) we did not have fancies like Jet, Mirus, Lipofectamine and so on. So, I took half a kilo jars with CaPO4, different mol weights PEI, prepared solutions and made titration transfections. 30-40 000 PEI was better than CaPO4 in vitro (

Dear All, Many thanks for your kind suggestions, but actually at the moment i am facing another problem, I tried two times without P/S but at both occasion cell got contaminated, thus the problem is that I can't do it without P/S. However what ever the protocol is all of them suggest to do without P/S, even I tried with OptiMEM either P/S just for 6 hrs, but in vain. 

Syed, you my want to re-precipitate your DNA with ethanol to reduce contamination.  Also, try this protocol:

Day 1 Plating (9-10am)

Plate 1-2x10^6 of 293T cells per 60-mm dish to have the next day 70-80% confluent monolayer.  Try several seeding densities to "nail" the optimal confluence

Day 2 Transfection (9-10am)

Replace medium with 4 ml of complete DMEM (+serum, antibiotics, etc).  Add chloroquin to 25 uM.

Prepare CaPO4 precipitate

In a sterile 1.5 ml tube mix the following:

Transfer vector - 20µg

Packaging plasmids - 20µg ( we use psPAX2 and pMD2.G. from Addgene)

Add 50µl 2.5M CaCl2 and water to 0.5ml.

Mix by vortexing

Add 0.5ml 2xHBS and mix well by vigorously pipetting up and down 6-8 times.

Keep in RT for 1-3 min, then add dropwise to a plate with your packaging cells and mix gently with a medium

Change medium (6-8hrs later); remove medium with precipitate and add fresh medium (5 ml/plate )

Day 3

Change medium again in early the morning

Collect your first "36h" supernatant just before you leave in the evening. Add fresh DMEM (5ml) to cells and leave plate in the CO2 incubator overnight. Filter your supernate through .45 um filter, aliquot, and freeze at -80°C

Day 4 Collection (9-10am)

Collect medium

Filter through 0.45 µm

At this point virus can be used for transduction, frozen at -80°C for future use, or concentrated

2 x HBS (for 500ml)

NaCl - 8g

KCl - 0.38g

Na2HPO4 - 0.1g

Hepes - 5g

Glucose - 1g Bring pH to 7.05 (important)

When performing transduction on your cells, do it in parallel on HeLa or other validated recipient to control for the possibility that your cells are reluctant to lentiviral transduction.

Many thanks Mikhail, 

I will let you know very soon how it goes.

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and making significant progress in your research endeavors, particularly in the realm of virology and gene therapy. Your inquiry regarding the efficiency of Lipofectamine 3000 compared to the calcium phosphate (CaPO4) transfection method for generating lentiviruses in 293FT cells is both timely and relevant, given the critical role of efficient viral vector production in therapeutic applications and research.

The generation of lentiviruses, especially in a cell line as widely used as 293FT, is a cornerstone technique in molecular biology and therapeutic research. The choice of transfection method can significantly impact the yield and quality of produced viruses, affecting downstream applications. Herein, I provide a comparative analysis based on available literature and empirical evidence:

Lipofectamine 3000

Lipofectamine 3000 is a more recent addition to the repertoire of transfection reagents, designed to enhance the efficiency of DNA delivery into a wide range of cell types, including difficult-to-transfect cells. Key advantages include:

• Higher Transfection Efficiency: Lipofectamine 3000 is formulated to provide higher transfection efficiencies compared to earlier versions and other transfection reagents. This is particularly advantageous for generating lentiviruses, where the quantity of transfected DNA directly correlates with viral yield.
• Lower Cytotoxicity: The formulation is optimized to reduce cytotoxic effects often associated with transfection, potentially leading to healthier cell cultures post-transfection and possibly higher viral titers.
• Versatility and Ease of Use: Designed for a broad range of applications, Lipofectamine 3000 simplifies the transfection process, requiring less optimization for different DNA types and cell lines.

Calcium Phosphate (CaPO4) Transfection

The CaPO4 transfection method is a long-standing, widely used technique for introducing DNA into mammalian cells, including 293FT cells. While cost-effective and efficient for many applications, it presents certain limitations in comparison to Lipofectamine 3000:

• Variable Efficiency: The efficiency of CaPO4 transfection can be highly variable, depending on the cell type and conditions. Precise pH and buffer conditions are critical for success, requiring careful optimization.
• Cytotoxicity: CaPO4 precipitation can be cytotoxic to cells, particularly at higher concentrations or with prolonged exposure. This may affect cell viability and, subsequently, viral production.
• Technical Sensitivity: The method is sensitive to various parameters, including DNA quality, pH of the solutions, and the timing of the precipitation process. Inconsistencies in these parameters can lead to variations in transfection efficiency.

Comparative Efficiency in Lentivirus Production

In the context of generating lentiviruses in 293FT cells, Lipofectamine 3000 generally demonstrates higher efficiency and reliability compared to the CaPO4 method. This is attributed to its higher transfection efficiency, reduced cytotoxicity, and greater ease of use. However, the CaPO4 method remains a cost-effective alternative for labs with the expertise to optimize and handle its variability.

It is important to note that the ultimate choice between Lipofectamine 3000 and CaPO4 may also depend on specific experimental needs, budget constraints, and available expertise. Laboratories may choose to validate both methods side-by-side for their specific application to determine the most effective approach for their needs.

I trust this comparison provides a comprehensive overview to assist in your decision-making process. Should you have further questions or require additional insights, please do not hesitate to reach out.

Warm regards.

Take a look at this protocol list; it could assist in understanding and solving the problem.