Frequently Asked Questions | In Vivo Delivery

pLIVE® In Vivo Vectors

The pLIVE® Vector (Liver In Vivo Expression) is designed for high level, prolonged expression of transgenes in the mouse liver. This vector utilizes a chimeric promoter composed of the mouse alpha fetoprotein enhancer II and the minimal mouse albumin promoter. Two introns have been engineered into the vector to increase the expression of the delivered transgene. Downstream of the first intron is a multiple cloning site (MCS) with eight unique restriction sites allowing for simple insertion of the gene of interest. Together the chimeric promoter and two introns are capable of promoting high level transgene expression in the liver for extended lengths of time compared to classic promoters such as the CMV immediate early promoter.

Two reporter vectors derived from pLIVE®, pLIVE®-lacZ (encoding β-galactosidase) and pLIVE®-SEAP (encoding Secreted Embryonic Alkaline Phosphatase), were created for use as positive controls. Expression of the lacZ gene from pLIVE®-lacZ can be monitored in the liver using either classical X-gal staining of liver sections or quantitative β-galactosidase assays of liver lysates. Expression of the SEAP gene from pLIVE®-SEAP can be easily monitored using a quantitative assay of mouse serum.

GENERAL QUESTIONS & ANSWERS

Q1. What elements are present in the pLIVE® Vector chimeric promoter?
Q2. How do expression levels compare between the pLIVE® Vectors and vectors utilizing the cytomegalovirus immediate early promoter (CMV promoter) to drive transgene expression?
Q3. How long will expression from the pLIVE® Vectors continue after delivery to the liver?
Q4. What is the key factor that affects the duration of transgene expression from the pLIVE® Vector?
Q5. How can the duration of transgene expression be increased?
Q6. Will the pLIVE® Vectors function in rats?
Q7. Can the pLIVE® Vectors be used in co-delivery applications for RNAi experiments?
Q8. Can two pLIVE® Vectors expressing different transgenes be delivered simultaneously to the mouse liver?

PROTOCOL QUESTIONS & ANSWERS

Q9. What is the E. coli selectable marker on the pLIVE® Vectors?
Q10. Are the pLIVE® Vectors high or low copy number plasmids?
Q11. What are the stability and storage conditions for the pLIVE® Vectors?
Q12. What is the best method for preparing pLIVE® Vector DNA before delivery?
Q13. What level of endotoxin in the pLIVE® Vector DNA is acceptable for in vivo delivery?
Q14. Which method does Mirus Bio recommend for delivery to the mouse liver?
Q15. Are the pLIVE® Vectors compatible with other delivery methods?
Q16. How much vector DNA should be delivered to each mouse?
Q17. Will the presence of an intron downstream of the pLIVE® MCS induce nonsense mediated decay (NMD) of the transgene transcript?
Q18. Is the sequence surrounding the transgene start codon important for optimal translation?
Q19. How can the expression of the lacZ reporter gene be assessed after delivery?
Q20. How should the liver lysates be treated when using the Galacto-Light™ Kit (Applied Biosystems) to quantify lacZ expression?
Q21. How can the expression of the SEAP reporter gene be assessed after delivery?
Q22. How should the serum samples be treated when assaying for SEAP activity?

TROUBLESHOOTING QUESTIONS & ANSWERS

Q23. I am not seeing any expression of the target transgene after delivery. What could be causing this problem?

GENERAL QUESTIONS & ANSWERS

Q1. What elements are present in the pLIVE® Vector chimeric promoter?
The chimeric promoter in the pLIVE® Vectors contains two important elements necessary for long-term, stable expression of transgenes in the liver. The first element is the mouse minimal albumin promoter, and the second element is the mouse alpha fetoprotein II enhancer.

Q2. How do expression levels compare between the pLIVE® Vectors and vectors utilizing the cytomegalovirus immediate early promoter (CMV promoter) to drive transgene expression?
We have compared the expression of the human placental secreted alkaline phosphatase gene (SEAP) from the pLIVE® Vector and a vector expressing the same reporter using the CMV promoter. Maximal expression from the CMV promoter is approximately 2.5 fold greater than the maximal expression achieved from the pLIVE®-SEAP Vector. However, expression from the CMV promoter rapidly decreases after delivery, and SEAP activity is negligible 10 days post-delivery. SEAP expression from the pLIVE® Vector increases from day 1 to day 4 post-injection and maintains that expression level for at least 8 months post-delivery.

Q3. How long will expression from the pLIVE® Vectors continue after delivery to the liver?
We have detected SEAP expression out to 8 months post-injection in mice.

Q4. What is the key factor that affects the duration of transgene expression from the pLIVE® Vector?
The key factor affecting duration of transgene expression is the immune response mounted by the mouse. If the protein expressed from the delivered transgene is immunogenic in the mouse, then the immune system will begin to destroy the liver cells expressing the transgene. An immune response to the transgene product will normally be evident by a rapid decrease in transgene expression 10-14 days post-injection.

Q5. How can the duration of transgene expression be increased?
The strain of mice used can have a dramatic effect on the duration of expression. In general, we have observed longer expression from the pLIVE® Vectors when using C57Bl/6 mice compared to ICR mice. In addition, the more similar the transgene product is to its mouse counterpart, the longer the predicted duration of expression. Finally, if a mouse homolog exists for the transgene of interest, expressing that mouse homolog in place of the current transgene should prevent an immune response from occurring, thus enabling maximal long-term expression.

Q6. Will the pLIVE® Vectors function in rats?
The chimeric promoter in the pLIVE® Vectors efficiently transcribes the transgene when delivered to the rat liver. If delivering the pLIVE® Vectors to the rat liver by hydrodynamic tail vein injection, we recommend increasing the amount of pLIVE® Vector DNA to 100-500 μg per injection.

Q7. Can the pLIVE® Vectors be used in co-delivery applications for RNAi experiments?
Yes. These vectors can be co-delivered with siRNA to achieve RNAi-mediated gene knockdown of the target gene expressed from the pLIVE® Vector.

Q8. Can two pLIVE® Vectors expressing different transgenes be delivered simultaneously to the mouse liver?
Yes. However, the level of expression of each transgene would be lower over time.

PROTOCOL QUESTIONS & ANSWERS

Q9. What is the E. coli selectable marker on the pLIVE® Vectors?
All the pLIVE® Vectors carry the kanamycin resistance gene, and E. coli cells harboring these plasmids should be grown in media containing 30 μg/ml of kanamycin.

Q10. Are the pLIVE® Vectors high or low copy number plasmids?
The plasmid backbone in the pLIVE® vectors is derived from the pUC vector, making them high copy number plasmids.

Q11. What are the stability and storage conditions for the pLIVE® Vectors?
The vectors are supplied in a low TE (Tris-EDTA) solution composed of 10 mM Tris, pH 7.5 and 0.1 mM EDTA. Store the vectors at -20°C and thaw at room temperature before use. These vectors are stable for at least 1 year from date of purchase if stored properly.

Q12. What is the best method for preparing pLIVE® Vector DNA before delivery?
We recommend using high purity plasmid purification kits that include an endotoxin removal step to purify DNA before delivery to mice. Residual endotoxin can also be removed using the MiraCLEAN® Endotoxin Removal Kit.

Q13. What level of endotoxin in the pLIVE® Vector DNA is acceptable for in vivo delivery?
Less than 30 Endotoxin Units/mg of DNA is acceptable. The presence of more endotoxin in the injected DNA could lead to an adverse reaction in the mice after delivery.

Q14. Which method does Mirus Bio recommend for delivery to the mouse liver?
We recommend using the hydrodynamic tail vein injection procedure for the delivery of nucleic acids to the mouse liver. We offer the TransIT®-QR and TransIT®-EE Hydrodynamic Delivery Solution and Kits for this purpose.

Q15. Are the pLIVE® Vectors compatible with other delivery methods?
Hydrodynamic tail vein injection is currently the most efficient non-viral method for the delivery of nucleic acids to the liver. However, any method that can successfully deliver DNA to the liver will result in expression from the pLIVE® Vectors.

Q16. How much vector DNA should be delivered to each mouse?
When using hydrodynamic injections to deliver the pLIVE® Vector DNA to the mouse liver, 10-50 μg of injected vector DNA is generally sufficient to obtain good transgene expression. However, the amount of DNA injected for a given experiment should be tested empirically to obtain the desired level of transgene expression. If using other delivery techniques, the amount of DNA will need to be determined empirically. For hydrodynamic tail vein delivery to rats, inject 100-500 μg of vector DNA.

Q17. Will the presence of an intron downstream of the pLIVE® MCS induce nonsense mediated decay (NMD) of the transgene transcript?
In general, it is believed that if the stop codon of an open reading frame is more than 50 bases upstream of the last exon-exon junction, then this transcript will be degraded by the cell, a process known as nonsense mediated decay (NMD) (1,2). If a transgene open reading frame is cloned into the pLIVE® Vector MCS such that the stop codon is adjacent to the Sac I, Sac II or Xho I sites, the stop codon will be less than 50 bases upstream of the last exon-exon junction preventing NMD. However, if a transgene open reading frame is cloned into the pLIVE® Vector MCS such that the stop codon is adjacent to the Nhe I, Asc I , Sal I, Sma I or BamH I sites, the stop codon will be more than 50 bases upstream of the last exon-exon junction increasing the likelihood of NMD. We have cloned the firefly luciferase open reading frame into the pLIVE® Vector such that the stop codon is adjacent to either Nhe I, Sal I, BamH I, or Xho I sites. Maximal expression was seen when the stop codon was adjacent to the Xho I site, and was approximately 2-3 times greater than the expression observed from the other clones, suggesting that NMD can occur. However, luciferase expression from the Nhe I clone was still high and easily detected.

Q18. Is the sequence surrounding the transgene start codon important for optimal translation?
Yes. When cloning the gene of interest (GOI) fragment into the MCS of the pLIVE® Vector, design the sequence around the ATG start codon (underlined) such that it matches the Kozak translational consensus sequence: (G/A)NNATGG (3,4). This consensus sequence will promote the optimal translation efficiency of the GOI mRNA.

Q19. How can the expression of the lacZ reporter gene be assessed after delivery?
After delivery of the pLIVE®-lacZ Vector, the livers can either be harvested, sectioned and stained with X-gal to stain the lacZ expressing cells blue, or the livers can be homogenized and the lysates can be assayed for β-galactosidase activity using a quantitative assay. For the quantitative assay of liver lysates, we recommend using the chemiluminescent Galacto-Light™ Kit from Applied Biosystems.

Q20. How should the liver lysates be treated when using the Galacto-Light™ Kit (Applied Biosystems) to quantify lacZ expression?
After harvesting the livers, we recommend lysing each liver sample in approximately 4 ml of a standard lysis buffer (not supplied), and then dilute 100-500 fold before assaying according to the kit’s protocol.

Q21. How can the expression of the SEAP reporter gene be assessed after delivery?
After delivery of the pLIVE®-SEAP Vector, the serum can be harvested and assayed for SEAP activity using a quantitative assay such as the Phospha-Light™ Kit from Applied Biosystems.

Q22. How should the serum samples be treated when assaying for SEAP activity?
Depending on the delivery efficiency and expression level of the pLIVE®-SEAP Vector, we recommend diluting the serum samples 100-500 fold before assaying using the Phospha-Light™ Kit (Applied Biosystems). This level of dilution normally produces SEAP activities that are within the useful range of the standard curve generated using purified SEAP protein.

TROUBLESHOOTING QUESTIONS & ANSWERS

Q23. I am not seeing any expression of the target transgene after delivery. What could be causing this problem?
Poor expression of the target transgene can be caused due to the following reasons:

1. Improper delivery method – It is possible that the delivery method being used is not effectively delivering the vectors to the liver. If using hydrodynamic tail vein injections, use a nucleic acid solution equivalent to 1/10 the volume of the mouse with 10 μg of vector DNA. It is critical to administer the entire solution into the tail vein in one smooth injection over 4-7 seconds.
2. Age of the mice – Use young, approximately 20 g mice for hydrodynamic tail vein injections. Larger mice generally produce lower and less reproducible levels of transgene expression.
3. Suboptimal sequence surrounding the ATG start codon of the transgene – Design the sequence surrounding the ATG start codon (underlined) to have the following sequence: (G/A)NNATGG (3,4). This sequence should promote optimal translation efficiency.
4. Suboptimal location of the transgene stop codon in the pLIVE® Vector MCS – If the stop codon of the transgene open reading frame is more than 50 bases upstream of the 5’ end of intron 2 (base 988), nonsense mediated decay could be induced in the cell, leading to a decreased level of the transgene mRNA in the cell (1,2). We have seen a 2-3 fold decrease in the level of luciferase expression when the luciferase stop codon is more than 50 bp upstream of the start of intron 2. However, we have not observed a total absence of transgene expression when the stop codon is more than 50 bases upstream of intron 2.
5. The protein product expressed from the transgene induces an immune response – If the transgene protein is immunogenic in mice, it will induce an immune response that will ultimately lead to clearance of the liver cells expressing the transgene. An immune response will usually take 10-14 days to develop and clear the cells. Harvest the livers and assay for transgene expression at an earlier timepoint. Alternatively, use C57Bl/6 mice which usually develop less robust immune responses compared to strains such as ICR.

References

1. Killing the messenger: new insights into nonsense-mediated mRNA decay. Byers, P.H., J. Clin. Invest.(2002) 109:3-6.
2. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Baker, K.E. and R. Parker, Curr. Opin. Cell Biol. (2004) 16:293-299.
3. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Kozak, M. Nuc. Acids Res.(1987) 15:8125-8148.
4. An analysis of vertebrate mRNA sequences: intimations of translational control. Kozak, M., J. Cell Biol. (1991) 115:887-903.