Hi Charitha,

You can definitely use the lentiviral construct for transfection and perform antibiotic selection. We always use LipoGeneTM Transfection Reagent, it shows very high transfection efficiency. You can find detailed information in the following website: https://www.genemedi.net/i/lipogene-transfection-reagent

The gene of your interest can be subcloned to a PEGFP/ promoter LUC construct by the method of Restriction enzyme digestion/ligation or the method of Gibson assembly. If you choose Gibson assembly, ClonEasyTM One Step Cloning Kit in Genemedi with high transformation efficiencies is a good choice for you. You can find detailed information in the following website: https://www.genemedi.net/i/cloneasy-one-step-cloning-kit

Hope this may help you.

Hello

you can use Lipofectamine® LTX ( by life technologies ) procedure to transfect plasmid DNA into MCF-7 cells in a 24-well format (for other formats, to know Scaling Up or Down Transfections, see the links below). All amounts and volumes are given on a per well basis. follow this Protocol :

1. The day before transfection, trypsinize and count the cells. Plate 4 x 104 cells per well in 0.5 ml of complete growth medium. Cell density should be 50~80% confluent on the day of transfection.

2. For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum.

3. For each well of cells, dilute 1.25-2.25 μl of Lipofectamine® LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine® LTX complexes.

4. Remove growth medium from cells and replace with 0.5 ml of complete growth medium. Add 100 μl of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.

5. Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression.

- Determination of viral titre allows subsequent lentiviral infection at intended infection efficiencies, using informed dilutions of lentivirus (e.g. MCF7 cells ) .

1. The day before transduction, count 293T with the cell counter and seed 24 well plates with target cell line at 5x104 cells per well. Plate extra wells so that at the time of transduction, the cells from one well can be counted for a more accurate cell number.

2. After 24h, count the cells in one 24-well.

3. Prepare the 1 in 3 serial dilutions of the virus in media.

4. Remove the media from the 24 well plates and add the 400ul virus dilutions to each required well starting with 400ul of neat virus. Repeat for 5 dilutions.

5. Incubate for 24h at 37°C, 5% CO2. Replace the virus with 1ml fresh media.

6. Incubate for a further 48h at 37°C, 5% CO2. Measure the %GFP by FACS for each of the wells counting 5000 events.

7. Calculate the titre expressed as transducing units (TU)/ml

Transducing units = (F x C/V) x D

F= frequencing of GFP positive cells (% divided by 100)

C= total number of cells in well at time of transduction

V= volume of inoculum in ml (400ul = 0.4ml)

D= lentivirus (dilution).

NOT:

To transfect MCF-7 cells in different tissue culture formats, vary the amounts of Lipofectamine® LTX Reagent, DNA, cells, medium and PLUS™ Reagent used in proportion to the relative surface area .

References as links . Good Luck

http://www.lifetechnologies.com/sa/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-mcf-7-cells-using-lipofectamine-ltx-reagent.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934029/

With out amplification of virus in 293t cells , can I directly transfect into mcf the same plasmid.? The promoter is PCMV. Or amplification can be done in bacteria like PEGFP ?

You can very well use these lentiviral vectors as plasmid DNA after transformation into any bacteria and transfect MCF-7 cells using any transfection agent.If you want to use this as a viral aprticle,you need 293t cells with packaging extract etc,but here you dont use any transfection reagent.Viral particles show high level of transduction.

Hi Charitha,

Yes you can use these vectors directly as expression vectors, the only caveat to this is they are large and may not transfect or persist as well as a dedicated expression vector.  It is commen to test lentiviral constructs by transfection before putting the effort in to package them up.  Without packaging you may get some expression from the 5ltr of the viral promoter, as it is not inactivated until packaging, this leads to complications if you are using cell specific promoters, but as you are using a ubiquitous high level viral promoter (PCMV) it should not be a problem.

You also mention antibiotic selection, if you wish to make a cell line, then either transfer the reporter into an expression plasmid of choice or (if the lentivirus has antibiotic selection) package it up into viral particles (providing you have the correct facilities and permissions to do this) and use that.  It is a very effective way of quickly establishing stable cell lines

It is our routine to test lentiviral constructs we build by transfecting them without other plasmids needed for virus production. I completely agree with David. Do as David said, It will work.

Bests

Hi Charitha,

I guess lots of people agree it will work, I just forgot to mention one thing.  If you are growing up the lentiviral constructs to get large ammounts of plasmid just to transfect, it is still  important to still use E.coli cells cabable of cloning direct repeats like those found in letivirus, something like StBL3's as otherwise you risk recombining out bits of the plasmid

good luck with your experiments

Thank  you all for the valuble inputs given and the time you took to answer my question

Hi Charitha,

You can definitely use the lentiviral construct for transfection and perform antibiotic selection. We always use LipoGeneTM Transfection Reagent, it shows very high transfection efficiency. You can find detailed information in the following website: https://www.genemedi.net/i/lipogene-transfection-reagent

The gene of your interest can be subcloned to a PEGFP/ promoter LUC construct by the method of Restriction enzyme digestion/ligation or the method of Gibson assembly. If you choose Gibson assembly, ClonEasyTM One Step Cloning Kit in Genemedi with high transformation efficiencies is a good choice for you. You can find detailed information in the following website: https://www.genemedi.net/i/cloneasy-one-step-cloning-kit

Hope this may help you.

Can I then transfect the antibiotic-selected cell line with the packaging constructs to get infectious virions?