Titering lentiviral vectors

Comparison of DNA, RNA and marker expression methods

L. Sastry, T. Johnson, M. J. Hobson, B. Smucker, Kenneth Cornetta

Research output: Contribution to journalArticle

210 Citations (Scopus)

Abstract

To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 ± 0.25 × 106 molecules/ml at 14 days, and 2.31 ± 0.15 × 106 molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 ± 0.04 × 106 TU/ml at 14 days, and 0.24 ± 0.03 ± 106 TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (∼ 103-fold) and GFP titers (∼ 104-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 ± 1.4 × 105 molecules/ml), and SMPU-RRE-BN (1.26 ± 0.2 × 106 molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.

Original languageEnglish
Pages (from-to)1155-1162
Number of pages8
JournalGene Therapy
Volume9
Issue number17
DOIs
StatePublished - Sep 2002

Fingerprint

Genetic Markers
RNA
DNA
Real-Time Polymerase Chain Reaction
Plasmids
Defective Viruses
Product Packaging
Transgenes
Fluorescence
Cell Line
Polymerase Chain Reaction

Keywords

  • DNA titer
  • GFP titer
  • Lentiviral vectors
  • Packaging probe
  • Real-time PCR
  • RNA titer

ASJC Scopus subject areas

  • Genetics

Cite this

Titering lentiviral vectors : Comparison of DNA, RNA and marker expression methods. / Sastry, L.; Johnson, T.; Hobson, M. J.; Smucker, B.; Cornetta, Kenneth.

In: Gene Therapy, Vol. 9, No. 17, 09.2002, p. 1155-1162.

Research output: Contribution to journalArticle

Sastry, L. ; Johnson, T. ; Hobson, M. J. ; Smucker, B. ; Cornetta, Kenneth. / Titering lentiviral vectors : Comparison of DNA, RNA and marker expression methods. In: Gene Therapy. 2002 ; Vol. 9, No. 17. pp. 1155-1162.
@article{c5af28c81d8041b9a29561fc1bae483d,
title = "Titering lentiviral vectors: Comparison of DNA, RNA and marker expression methods",
abstract = "To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 ± 0.25 × 106 molecules/ml at 14 days, and 2.31 ± 0.15 × 106 molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 ± 0.04 × 106 TU/ml at 14 days, and 0.24 ± 0.03 ± 106 TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (∼ 103-fold) and GFP titers (∼ 104-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 ± 1.4 × 105 molecules/ml), and SMPU-RRE-BN (1.26 ± 0.2 × 106 molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.",
keywords = "DNA titer, GFP titer, Lentiviral vectors, Packaging probe, Real-time PCR, RNA titer",
author = "L. Sastry and T. Johnson and Hobson, {M. J.} and B. Smucker and Kenneth Cornetta",
year = "2002",
month = "9",
doi = "10.1038/sj.gt.3301731",
language = "English",
volume = "9",
pages = "1155--1162",
journal = "Gene Therapy",
issn = "0969-7128",
publisher = "Nature Publishing Group",
number = "17",

}

TY - JOUR

T1 - Titering lentiviral vectors

T2 - Comparison of DNA, RNA and marker expression methods

AU - Sastry, L.

AU - Johnson, T.

AU - Hobson, M. J.

AU - Smucker, B.

AU - Cornetta, Kenneth

PY - 2002/9

Y1 - 2002/9

N2 - To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 ± 0.25 × 106 molecules/ml at 14 days, and 2.31 ± 0.15 × 106 molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 ± 0.04 × 106 TU/ml at 14 days, and 0.24 ± 0.03 ± 106 TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (∼ 103-fold) and GFP titers (∼ 104-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 ± 1.4 × 105 molecules/ml), and SMPU-RRE-BN (1.26 ± 0.2 × 106 molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.

AB - To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 ± 0.25 × 106 molecules/ml at 14 days, and 2.31 ± 0.15 × 106 molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 ± 0.04 × 106 TU/ml at 14 days, and 0.24 ± 0.03 ± 106 TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (∼ 103-fold) and GFP titers (∼ 104-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 ± 1.4 × 105 molecules/ml), and SMPU-RRE-BN (1.26 ± 0.2 × 106 molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.

KW - DNA titer

KW - GFP titer

KW - Lentiviral vectors

KW - Packaging probe

KW - Real-time PCR

KW - RNA titer

UR - http://www.scopus.com/inward/record.url?scp=0036713630&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036713630&partnerID=8YFLogxK

U2 - 10.1038/sj.gt.3301731

DO - 10.1038/sj.gt.3301731

M3 - Article

VL - 9

SP - 1155

EP - 1162

JO - Gene Therapy

JF - Gene Therapy

SN - 0969-7128

IS - 17

ER -