Nonviral transfection of intact pancreatic islets

J. R T Lakey, A. T L Young, D. Pardue, S. Calvin, Timothy E Albertson, L. Jacobson, T. J. Cavanagh

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-β-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 μl) and DNA (3 and 6 μg). Transfection efficiency was quantified by microscopic evaluation of β-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express β-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 μl DNA and 18 μl DOTAP/ml (1:6 ratio), 6 μg DNA and 12 μl DOSPER/ml (1:2 ratio), or 6 μg DNA and 12 μg LipofectAMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8±0.6 (mean±SEM) for DOTAP-transfected islets compared with 8.4±0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1±0.5 and 3.4± 0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 Provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

Original languageEnglish (US)
Pages (from-to)697-708
Number of pages12
JournalCell Transplantation
Volume10
Issue number8
StatePublished - 2001
Externally publishedYes

Fingerprint

Islets of Langerhans
Transfection
DNA
Galactosidases
Lipids
Liposomes
Insulin
Grafts
Genes
Gene transfer
Transplantation Tolerance
Graft Rejection
Analysis of variance (ANOVA)
Gene expression
Glucose
Toxicity
1,2-dioleoyloxy-3-(trimethylammonium)propane
Analysis of Variance
Plasmids
Gene Expression

Keywords

  • Diabetes
  • Islets
  • Liposomes
  • Transfection
  • Transplantation

ASJC Scopus subject areas

  • Cell Biology
  • Transplantation

Cite this

Lakey, J. R. T., Young, A. T. L., Pardue, D., Calvin, S., Albertson, T. E., Jacobson, L., & Cavanagh, T. J. (2001). Nonviral transfection of intact pancreatic islets. Cell Transplantation, 10(8), 697-708.

Nonviral transfection of intact pancreatic islets. / Lakey, J. R T; Young, A. T L; Pardue, D.; Calvin, S.; Albertson, Timothy E; Jacobson, L.; Cavanagh, T. J.

In: Cell Transplantation, Vol. 10, No. 8, 2001, p. 697-708.

Research output: Contribution to journalArticle

Lakey, JRT, Young, ATL, Pardue, D, Calvin, S, Albertson, TE, Jacobson, L & Cavanagh, TJ 2001, 'Nonviral transfection of intact pancreatic islets', Cell Transplantation, vol. 10, no. 8, pp. 697-708.
Lakey JRT, Young ATL, Pardue D, Calvin S, Albertson TE, Jacobson L et al. Nonviral transfection of intact pancreatic islets. Cell Transplantation. 2001;10(8):697-708.
Lakey, J. R T ; Young, A. T L ; Pardue, D. ; Calvin, S. ; Albertson, Timothy E ; Jacobson, L. ; Cavanagh, T. J. / Nonviral transfection of intact pancreatic islets. In: Cell Transplantation. 2001 ; Vol. 10, No. 8. pp. 697-708.
@article{6941c7f198fc4a0cb38faedd3ff06fe3,
title = "Nonviral transfection of intact pancreatic islets",
abstract = "Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-β-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 μl) and DNA (3 and 6 μg). Transfection efficiency was quantified by microscopic evaluation of β-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express β-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 μl DNA and 18 μl DOTAP/ml (1:6 ratio), 6 μg DNA and 12 μl DOSPER/ml (1:2 ratio), or 6 μg DNA and 12 μg LipofectAMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8±0.6 (mean±SEM) for DOTAP-transfected islets compared with 8.4±0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1±0.5 and 3.4± 0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 Provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.",
keywords = "Diabetes, Islets, Liposomes, Transfection, Transplantation",
author = "Lakey, {J. R T} and Young, {A. T L} and D. Pardue and S. Calvin and Albertson, {Timothy E} and L. Jacobson and Cavanagh, {T. J.}",
year = "2001",
language = "English (US)",
volume = "10",
pages = "697--708",
journal = "Cell Transplantation",
issn = "0963-6897",
publisher = "Cognizant Communication Corporation",
number = "8",

}

TY - JOUR

T1 - Nonviral transfection of intact pancreatic islets

AU - Lakey, J. R T

AU - Young, A. T L

AU - Pardue, D.

AU - Calvin, S.

AU - Albertson, Timothy E

AU - Jacobson, L.

AU - Cavanagh, T. J.

PY - 2001

Y1 - 2001

N2 - Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-β-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 μl) and DNA (3 and 6 μg). Transfection efficiency was quantified by microscopic evaluation of β-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express β-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 μl DNA and 18 μl DOTAP/ml (1:6 ratio), 6 μg DNA and 12 μl DOSPER/ml (1:2 ratio), or 6 μg DNA and 12 μg LipofectAMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8±0.6 (mean±SEM) for DOTAP-transfected islets compared with 8.4±0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1±0.5 and 3.4± 0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 Provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

AB - Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-β-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 μl) and DNA (3 and 6 μg). Transfection efficiency was quantified by microscopic evaluation of β-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express β-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 μl DNA and 18 μl DOTAP/ml (1:6 ratio), 6 μg DNA and 12 μl DOSPER/ml (1:2 ratio), or 6 μg DNA and 12 μg LipofectAMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8±0.6 (mean±SEM) for DOTAP-transfected islets compared with 8.4±0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1±0.5 and 3.4± 0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 Provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

KW - Diabetes

KW - Islets

KW - Liposomes

KW - Transfection

KW - Transplantation

UR - http://www.scopus.com/inward/record.url?scp=0035696399&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035696399&partnerID=8YFLogxK

M3 - Article

VL - 10

SP - 697

EP - 708

JO - Cell Transplantation

JF - Cell Transplantation

SN - 0963-6897

IS - 8

ER -