3t3 is one of the easier cells to infect. I would first investigate whether your lentivirus vector is working well. Try it in other cells (HEK293s, HeLas). Which lentivirus pseudotype are you working with? Is your alternative a gamma retrovirus? If so, which pseudotype for that too?

I agree with Simon that 3T3 is one of the easiest cell to infect. In fact, we use 3T3 to titre the retro/lentiviral sup. For MigR1 retroviral backbone, I have achieved up to 80% GFP+ (measured by flow cytometry). You need to troubleshoot whether the problem of low GFP expression is due to transduction of 3T3, the way you make the viral particles or the validity of the expression vector.

Do a simple chemical (eg. xtremegene, turbofect, Calcium phosphate etc) transfection with 3T3 cells using the expression vector directly (without the packaging plasmids). If you see high GFP expression, then you need to optimize the way you make viral particles by testing different packaging plasmids. Some articles do mention in their methods which packaging plasmids work well with the specific expression vector.

3T3s don't give good expression when you're using human promoters such as hCMV or hEF1a (see picture here under Supporting Data - http://dharmacon.gelifesciences.com/shrna/smartchoice-shrna-promoter-selection-plate/). So your infection might be working just fine but expression levels could be too low to detect.

Hey thanks for your suggestion!

I made lentivirus (packaging: VSVG, REV,pDNL) and retrovirus (pSER-GFP)using 1:5 DNA:PEI ratio in 293T. The GFP expression in 293T is close to 90% but the harvested virus infected in 3t3 cells does not give any GFP signal. 

Can anyone share a protocol for the infection of mouse 3t3 cells or any research paper that someone has come across.

Thanks

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and making significant progress in your cell biology or virology research. Your inquiry regarding suitable viruses for infecting mouse 3T3 fibroblasts is both relevant and critical for designing experiments aimed at understanding virus-host interactions, viral lifecycles, or for utilizing viral vectors in gene delivery systems. Below, I detail a selection of viruses that have been successfully used to infect mouse 3T3 fibroblast cells, considering their applications and implications for research.

Viruses Suitable for Infecting Mouse 3T3 Fibroblasts

Best Practices and Considerations

• Biosafety: Adhere to appropriate biosafety protocols and containment levels when handling and disposing of viral materials, considering the pathogenicity of the virus and the potential for recombinant vector production.
• Viral Titer Optimization: Optimize the multiplicity of infection (MOI) to balance efficient cell transduction with minimal cytotoxicity.
• Experimental Controls: Include mock-infected and uninfected controls in your experiments to accurately assess viral infection effects.
• Ethical and Regulatory Compliance: Ensure all experiments involving viral vectors comply with institutional, local, and national guidelines on genetic manipulation and biosafety.

Conclusion

Selecting the appropriate virus for infecting mouse 3T3 fibroblasts depends on the specific objectives of your research, including whether the goal is gene delivery, studies of viral replication, or investigation of cellular responses to viral infection. By considering the characteristics and applications of each virus, as well as adhering to best practices in virology research, you can effectively design and conduct your experiments.

Should you require further guidance or wish to discuss the specific aspects of working with these viral systems, please do not hesitate to reach out. I am here to support your research efforts and facilitate the advancement of your scientific inquiries.

Warm regards.

This protocol list might provide further insights to address this issue.