I am trying to infect mouse embryonic stem cell(Ezh2-/-) with pLVX-ef1 alpha-ires-puro vector from clontech. I am using second generation packaging virus for lentivirus production. I transfected them in 293t cells and collected the 48 and 72 hours supernatent and directly added them to the mouse es cells. I left them for 48 hours, I started the puro selection and followed it for a week. I had infected my empty plasmid and my plasmid with the insert. The empty plasmid infected the cells but the plasmids with the insert didn't seem to infect the cells. My plasmid size was around 8.6 kb and my insert size is 2.2 kb. I am not sure now where the problem relies. Is there any good packaging system that I opt for? Will ultracentrifugation fetch me good titre?
Hi Amanda Thompson,
Thanks for the reply. The Insert that I cloned in to the vector is Mouse Ezh2 and it have EF1a promoter as you referred. As far as I searched in literature they have successfully cloned and infected the gene in many vectors. Gene toxicity is ruled out and I did restriction digestion to check whether the puro was intact and it seems like it is intact. I am using VSV-G as envelope and I don't think i will have problems while doing ultra centrifugation. I have tried every way and I was unsuccessfully in these attempts to infect the mouse embryonic stem cells.
To me this looks like an issue related to the expression level of the Puro(R) gene, in "empty" vector (MCS-IRES-Puro) vs. gene-IRES-Puro vector. While IRES serves as a cap-independent translational initiation site, the distance to the 5'cap of the transcript still matters somehow. In our IRES-GFP lentiviral constructs, we always see higher average GFP levels in empty control vectors (MCS-IRES-GFP) than in constructs containing a first cistron (gene-IRES-GFP).
Translated into your IRES-Puro scenario, it might mean that the concentration of puromycin you are using to select for transduced ES cells is OK for empty vector, but too high for gene-containing vector - killing off the cells although they are transduced.
My suggestion is to try lower concentrations of puromycin. Find the lowest concentration that will reliably kill non-transduced cells, and start from there. Also, you can make a GFP-IRES-Puro control vector, which allows you to directly monitor the selectivity of your puromycin concentration.
Good luck!
Alex
Dear [Recipient],
I trust this message finds you well. I understand that you're seeking solutions for challenges encountered during lentivirus production and infection processes. Lentiviral vectors are indispensable tools in molecular biology, particularly for gene delivery and gene expression studies in both dividing and non-dividing cells. However, optimizing both the production and infection steps can be complex due to the intricate variables involved. Let's address some common issues and propose potential solutions.
1. Low Lentivirus Production Yield
Potential Causes:
- Inefficient transfection of packaging cells.
- Suboptimal packaging cell line health or density.
- Inadequate vector design.
Solutions:
- Enhance Transfection Efficiency: Utilize high-quality transfection reagents and optimize the DNA-to-reagent ratio. Ensure the plasmid DNA is of high purity.
- Cell Line Optimization: Maintain packaging cells under optimal conditions to ensure they are in a vigorous growth phase at the time of transfection. The cell confluency should ideally be around 70-80%.
- Vector Optimization: Review the vector design for any elements that might reduce viral production, including the size of the insert (ideally less than 5kb for optimal packaging efficiency) and the inclusion of appropriate viral packaging signals.
2. Low Infection Efficiency
Potential Causes:
- Low viral titer.
- Inappropriate target cell line conditions.
- Insufficient polybrene concentration, if used to enhance infection efficiency.
Solutions:
- Titer Enhancement: Concentrate the virus using ultracentrifugation or precipitation methods to increase the viral titer.
- Target Cell Optimization: Ensure that the target cells are healthy and at an appropriate density during infection. Cells too sparse or too confluent may display reduced infection efficiency.
- Polybrene Use: If not already used, consider incorporating polybrene into your infection protocol to neutralize charge interactions between the virus and the cell membrane, enhancing infection efficiency. The optimal polybrene concentration may vary between cell types and should be determined empirically.
3. Toxicity Post-Infection
Potential Causes:
- High polybrene concentration.
- Overexpression of the transgene.
Solutions:
- Adjust Polybrene Concentration: If toxicity is observed, reducing the polybrene concentration can mitigate cell death without significantly compromising infection efficiency.
- Control Transgene Expression: Utilize inducible promoters to control the level and timing of transgene expression, reducing cellular stress and toxicity.
Additional Tips
- Quality Control: Regularly performing viral titer quantification and assessing the functional transduction efficiency can provide insights into the process and highlight steps requiring optimization.
- Sterility: Ensure all processes are conducted under sterile conditions to prevent contamination, which can significantly impact virus production and infection efficiency.
In summary, addressing issues in lentivirus production and infection often requires a multifaceted approach, focusing on optimizing transfection conditions, cell health, vector design, and infection parameters. Each step should be carefully evaluated and optimized based on empirical evidence and specific experimental needs.
Should you require further assistance or wish to delve deeper into any of these suggestions, please do not hesitate to reach out. I am here to support your research endeavors.
Best regards,
Perhaps this protocol list can give us more information to help solve the problem.
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