Hi Caterina

Absolutely it is possible. I would recommend using a virus that is VSV-G pseudotyped (most commercially available ones are) as this will infect both mouse and rat (and also human if you want) cells with high efficiency. I'm not sure about neuron, but we've used them with great success to infect primary mouse and primary human mammary stem (and progenitor) cells.

Most of the big companies (and a myriad of smaller ones) sell lenti particles. And there are companies that can pretty much supply you with any human or mouse cDNA in lenti format (not sure about rat). Your biggest decision will be price and configuration. With regards to configuration you'll need to decide whether you would like a fluorescent protein with it (for FACS/fluorescence microscopy), or a drug selectable marker (eg Puro). Also, I should point out that the more you want to pack into your lenti the more difficult it will be to get reasonable titres of the virus (which is what you'll need for infecting primary rat/mouse cells). This applies particularly to inducible vectors which generally are very large (eg pLOC from openbiosystems, or pInducer).

Finally, a few tips:

- tet inducible vectors (especially tet3R) appear to be less leaky

- how a company titrates their lenti profoundly influences what you get: if possible go for a company that uses flow cytometry of infected cells as this measures functional virus (avoid p24 assay and qPCR of virus lysate as these are both indirect and vary wildly from vector to vector)

- if you get an option of using a viral-2A sequence between your gene of interest and your fluorescent reporter then I'd favour this option as the levels of your fluorescent reporter will be directly proportional to the amount of your gene of interest that was transcribed (all things being equal).

Hope this helps

Good luck

Al

You may want to try GeneCopoeia (www.genecopoeia.com). They may already have your mouse full length protein coding ORF cDNA clone in various vecrots or may be able to make one for you upon request. If they do not have your ORF cDNA of interest in a vector, they can insert it into various vectors from a cDNA library using their high fidelity propriatory technology. I do not work for GeneCopoeia but have used their service and products. I am sure other companies may be able to offer comparable service/products. I can only speak from my experience.

Hi Caterina

Absolutely it is possible. I would recommend using a virus that is VSV-G pseudotyped (most commercially available ones are) as this will infect both mouse and rat (and also human if you want) cells with high efficiency. I'm not sure about neuron, but we've used them with great success to infect primary mouse and primary human mammary stem (and progenitor) cells.

Most of the big companies (and a myriad of smaller ones) sell lenti particles. And there are companies that can pretty much supply you with any human or mouse cDNA in lenti format (not sure about rat). Your biggest decision will be price and configuration. With regards to configuration you'll need to decide whether you would like a fluorescent protein with it (for FACS/fluorescence microscopy), or a drug selectable marker (eg Puro). Also, I should point out that the more you want to pack into your lenti the more difficult it will be to get reasonable titres of the virus (which is what you'll need for infecting primary rat/mouse cells). This applies particularly to inducible vectors which generally are very large (eg pLOC from openbiosystems, or pInducer).

Finally, a few tips:

- tet inducible vectors (especially tet3R) appear to be less leaky

- how a company titrates their lenti profoundly influences what you get: if possible go for a company that uses flow cytometry of infected cells as this measures functional virus (avoid p24 assay and qPCR of virus lysate as these are both indirect and vary wildly from vector to vector)

- if you get an option of using a viral-2A sequence between your gene of interest and your fluorescent reporter then I'd favour this option as the levels of your fluorescent reporter will be directly proportional to the amount of your gene of interest that was transcribed (all things being equal).

Hope this helps

Good luck

Al

Hi Caterina,

You can always plan to do the same with a wide variety of suppliers present world wide.

It depends on your experimental mindset whether you would prefer to package your own viral particles using lentiviral packaging systems

I would personally recommend 3-4 suppliers listed below.

1) ABM Inc, USA www.abmgood.com (Cost effective and chance of getting a ready viral particle is high)

2) Origene Technologies Inc,

They have retroviral 2nd generation MMLV backbone vectors that can be packaged using a 2nd generation packaging system. Expression ready and validated.

3)Open Biosystems, Lafayette

Lentiviral backbone with 3rd generation vector plasmid. Trans lentiviral packaging system along with the clone.

4) Genocopoeia,

Lentiviral backbone and trans-lentiviral packaging both are available.

Hope the information helps you.

Best Wishes,

Gaurab

Yes, you can use lentivirus to stably transduce rodent primary cells. Be sure to determine which pseudotype (viral envelope) gives the best transduction efficiency on your cell line of interest. This might be best conducted using a marker gene first, such as GFP, luciferase, or some drug resistance gene (Puro or Neo). In my hands as mentioned above the VSVg envelope seems to have the best tropism and transduction efficiency and allows for the generation of high titer vector preps.

Some other things to consider. Does your primary cell divide in culture? Although lentiviral vectors do transduce non-dividing cells, the efficiency is not always as good as with dividing cells. High multiplicity of infections (MOI) may lead to multiple integrations and very high levels of gene expression. Depending on what gene you are using this may cause toxicity or may cause problems in interpreting results. If you can include a drug selectable marker in your final cassette it may be good to use a low MOI and select out transduced cells.

If you do not have the plasmids for generating lentiviral vectors you can check with the companies mentioned above or try and obtain plasmids from reserachers. Since you appear to be in Switzerland check out the following:

http://tronolab.epfl.ch/lentivectors

Dr. Trono was involved in the generation of the lentiviral vector system.

Best of luck,

David

Hi, you can transduce neurons with VSV pseudotyped lentivectors, you will need higher titers that for example astrocytes that will be transduced very well. You can have a look at this accepted manuscript I uploaded wich shows an in vitro and in vivo comparison between integration proficient and deficient lentivectors for the transduction of motor neurons, dorsal root ganglion neurons, astrocytes and microglial cells:

https://www.researchgate.net/publication/230807202_EFFICIENT_GENE_EXPRESSION_FROM_INTEGRATION-DEFICIENT_LENTIVIRAL_VECTORS_IN_THE_SPINAL_CORDPeluffo_Hugo_%28PhD%29*Foster_Edmund_%28PhD%29_Ahmed_Sherif_G._%28PhD%29_Lago_Natalia_%28PhD%29_Hutson_Thomas_H._%28PhD%29_Moon_Lawrence_%28PhD%29_Yip_Ping_%28PhD%29Wanisch_Klaus_%28PhD%29_Caraballo-Miralles_Victor_%28PhD%29_Olmos_Gabriel_%28PhD%29_Llad_Jernia_%28PhD%29_McMahon_Stephen_B._%28PhD%29_and_Yez-Muoz_Rafael_J._%28PhD%29_Accepted_Gene_Therapy

Good luck!

Yes Caterina! It is possible to transduce them. I transduce primary dermal stem cells or rodents with GFP and mCherry all the time and it works.

Many thanks to everyone.

I tried GeneCopoeia, and they put me through to LabOmics, their distributor in Europe.

Hi Caterina,

I don't know if Addgene supplies vectors/plasmids in Europe. I would recommend to take a look (http://www.addgene.org/), as most of constructs are very good and at a very affordable cost.