Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids

R. L. Walzem, M. A. Hickman, J. B. German, R. J. Hansen

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipidmediated transfection is poorly described. 3β[N-N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propanaminimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam®, Lipofectamine®, and Lipofectin®, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17β-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10% chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.

Original languageEnglish (US)
Pages (from-to)882-886
Number of pages5
JournalPoultry Science
Volume76
Issue number6
StatePublished - Jun 1997

Fingerprint

hepatoma
transfection
estrogens
luciferase
lipids
promoter regions
ethanolamine
cells
Photinus pyralis
DNA
Human herpesvirus 5
chickens
response elements
bromides
cultured cells
estradiol
plateaus
insulin
cell lines
cholesterol

Keywords

  • Avian hepatoma LMH-2A cells
  • Cationic lipid
  • Estrogen
  • Transient expression assay

ASJC Scopus subject areas

  • Animal Science and Zoology

Cite this

Walzem, R. L., Hickman, M. A., German, J. B., & Hansen, R. J. (1997). Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids. Poultry Science, 76(6), 882-886.

Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids. / Walzem, R. L.; Hickman, M. A.; German, J. B.; Hansen, R. J.

In: Poultry Science, Vol. 76, No. 6, 06.1997, p. 882-886.

Research output: Contribution to journalArticle

Walzem, RL, Hickman, MA, German, JB & Hansen, RJ 1997, 'Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids', Poultry Science, vol. 76, no. 6, pp. 882-886.
Walzem RL, Hickman MA, German JB, Hansen RJ. Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids. Poultry Science. 1997 Jun;76(6):882-886.
Walzem, R. L. ; Hickman, M. A. ; German, J. B. ; Hansen, R. J. / Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids. In: Poultry Science. 1997 ; Vol. 76, No. 6. pp. 882-886.
@article{2b7fc0dccfce4e1d9beff82b069a0e7d,
title = "Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids",
abstract = "LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipidmediated transfection is poorly described. 3β[N-N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propanaminimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam{\circledR}, Lipofectamine{\circledR}, and Lipofectin{\circledR}, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17β-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10{\%} chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.",
keywords = "Avian hepatoma LMH-2A cells, Cationic lipid, Estrogen, Transient expression assay",
author = "Walzem, {R. L.} and Hickman, {M. A.} and German, {J. B.} and Hansen, {R. J.}",
year = "1997",
month = "6",
language = "English (US)",
volume = "76",
pages = "882--886",
journal = "Poultry Science",
issn = "0032-5791",
publisher = "Poultry Science Association",
number = "6",

}

TY - JOUR

T1 - Transfection of Avian LMH-2A Hepatoma Cells with Cationic Lipids

AU - Walzem, R. L.

AU - Hickman, M. A.

AU - German, J. B.

AU - Hansen, R. J.

PY - 1997/6

Y1 - 1997/6

N2 - LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipidmediated transfection is poorly described. 3β[N-N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propanaminimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam®, Lipofectamine®, and Lipofectin®, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17β-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10% chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.

AB - LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipidmediated transfection is poorly described. 3β[N-N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propanaminimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam®, Lipofectamine®, and Lipofectin®, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17β-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10% chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.

KW - Avian hepatoma LMH-2A cells

KW - Cationic lipid

KW - Estrogen

KW - Transient expression assay

UR - http://www.scopus.com/inward/record.url?scp=0031156844&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031156844&partnerID=8YFLogxK

M3 - Article

C2 - 9181623

AN - SCOPUS:0031156844

VL - 76

SP - 882

EP - 886

JO - Poultry Science

JF - Poultry Science

SN - 0032-5791

IS - 6

ER -