GENETICALLY MODIFIED PRIMARY ASTROCYTES AS CELLULAR VEHICLES FOR GENE-THERAPY IN THE BRAIN

Citation
Ef. Lagamma et al., GENETICALLY MODIFIED PRIMARY ASTROCYTES AS CELLULAR VEHICLES FOR GENE-THERAPY IN THE BRAIN, Cell transplantation, 2(3), 1993, pp. 207-214
Citations number
40
Categorie Soggetti
Cytology & Histology
Journal title
ISSN journal
09636897
Volume
2
Issue
3
Year of publication
1993
Pages
207 - 214
Database
ISI
SICI code
0963-6897(1993)2:3<207:GMPAAC>2.0.ZU;2-Y
Abstract
Combining genetic engineering and cell transplantation has been propos ed as one way to overcome the limited availability of donor tissue tha t may restrict the application of graft therapy to neurological diseas es. Important issues in this approach concern the choice of a suitable cellular vehicle, and the method of gene insertion. In this regard, w e have investigated the use of brain-derived primary astrocytes as cel lular vehicles for gene therapy, because they can be transfected, divi de in culture, are brain-region specific, possess a secretory mechanis m, and may migrate several mm from the transplant site. To address the issue of gene insertion, we have generated stably transfected primary rat astrocytes using the nonviral calcium phosphate method to co-tran sfect a reporter construct (RSV-chloramphenicol acetyltransferase (CAT ), or human enkephalin promoter CAT, plus a neomycin resistance plasmi d (pRSVNeo). Modified astrocytes were then propagated by transfer to s elective media containing G418 (300 mug/mL) for 3 wk. The presence of the reporter gene product (CAT) was demonstrated by immunocytochemistr y, and by biochemical assay of CAT enzyme catalytic activity. These ge netically modified astrocytes were followed for up to 3 wk after trans plantation into the rat striatum. Criteria used to distinguish transpl anted astrocytes included histological evidence of abundant nuclei int errupting the normal cytoarchitecture of the striatum, astrocyte morph ology, and the presence of CAT enzyme activity. Our data indicates tha t genetically modified astrocytes are an important candidate vehicle f or use in transplantation therapy in neurological diseases. We suggest that genetically modified astrocytes can also be used for studying th e human enkephalin promoter, other promoters, and expressed proteins u sing this paradigm.