Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes

Dan Spandau, M. Marques, M. Bierhuizen, G. Wagemaker, S. Hurwitz, Y. Pei, R. Breese, Jeffrey Travers

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.

Original languageEnglish
Pages (from-to)252-257
Number of pages6
JournalExperimental Dermatology
Volume9
Issue number4
DOIs
StatePublished - Aug 2000

Fingerprint

Gene therapy
Keratinocytes
Genetic Therapy
Gene transfer
Cells
Foreskin
SCID Mice
Cell growth
Feasibility Studies
Etoposide
Retroviridae
Ultraviolet Rays
enhanced green fluorescent protein
Sorting
Heterografts
Epidermis
Genes
Purification
Light sources
Skin

Keywords

  • Apoptosis
  • Green fluorescent protein
  • Keratinocytes
  • Retrovirus

ASJC Scopus subject areas

  • Dermatology

Cite this

Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes. / Spandau, Dan; Marques, M.; Bierhuizen, M.; Wagemaker, G.; Hurwitz, S.; Pei, Y.; Breese, R.; Travers, Jeffrey.

In: Experimental Dermatology, Vol. 9, No. 4, 08.2000, p. 252-257.

Research output: Contribution to journalArticle

Spandau, Dan ; Marques, M. ; Bierhuizen, M. ; Wagemaker, G. ; Hurwitz, S. ; Pei, Y. ; Breese, R. ; Travers, Jeffrey. / Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes. In: Experimental Dermatology. 2000 ; Vol. 9, No. 4. pp. 252-257.
@article{7ad60ccbcdb74ed6abac2796c3beb025,
title = "Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes",
abstract = "Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.",
keywords = "Apoptosis, Green fluorescent protein, Keratinocytes, Retrovirus",
author = "Dan Spandau and M. Marques and M. Bierhuizen and G. Wagemaker and S. Hurwitz and Y. Pei and R. Breese and Jeffrey Travers",
year = "2000",
month = "8",
doi = "10.1034/j.1600-0625.2000.009004252.x",
language = "English",
volume = "9",
pages = "252--257",
journal = "Experimental Dermatology",
issn = "0906-6705",
publisher = "Wiley-Blackwell",
number = "4",

}

TY - JOUR

T1 - Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes

AU - Spandau, Dan

AU - Marques, M.

AU - Bierhuizen, M.

AU - Wagemaker, G.

AU - Hurwitz, S.

AU - Pei, Y.

AU - Breese, R.

AU - Travers, Jeffrey

PY - 2000/8

Y1 - 2000/8

N2 - Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.

AB - Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.

KW - Apoptosis

KW - Green fluorescent protein

KW - Keratinocytes

KW - Retrovirus

UR - http://www.scopus.com/inward/record.url?scp=0033909950&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033909950&partnerID=8YFLogxK

U2 - 10.1034/j.1600-0625.2000.009004252.x

DO - 10.1034/j.1600-0625.2000.009004252.x

M3 - Article

C2 - 10949546

AN - SCOPUS:0033909950

VL - 9

SP - 252

EP - 257

JO - Experimental Dermatology

JF - Experimental Dermatology

SN - 0906-6705

IS - 4

ER -