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
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.