Download Differentiation of a Schwann cell line expressing reporter genes in

Biochemical Society Transactions (1997) 25 5438
178 Differentiation of a sehwpnn cell line expressing reporter
genes in the presence and absence of nerve axons
ANTHONYR. FOOKS', SABINESCHMI-IZ', JAMES A. RUSHTON',
STEVEN J. HOWE', PAUL D. GRAHAM', VARSHA GODBOLE',
GLYN STACEY', J. CHRISTOPHER S. CLEGG' and LAURENCE W.
H A m Z
'Centre for Applied Microbiology and Research, Porton Down, Salisbury,
Wilts SP4 OJG and *School of Biological Sciences, University of Bristol,
Woodland Road,Bristol BS8 IUG
The formation of the myelin sheath in peripheral nerves requires a
complex sequence of morphological interactions between myelincompetent Schwann cells and the nerve axon and a concomitant switch in
Schwann cell gene expression patterns from the immature to the mature
myelin-forming phenotype [l]. The degree of interdependence between
these two phenomena is not yet understood. To explore links between
molecular and cellular events in myelination and demyelination we are
expressing reporter genes in clonallyderived Schwann cells in order to
study gene activity in relation to axon ensheathment in neuron coculture.
The SCL4.lk7 immortal diploid Schwann cell strain 121 can
undergo postmitotic differentiation in culture either in the presence or the
absence of neurons. Rapidly growing cells were transfected using CaZPO4
coprecipitation with plasmid pffiFP-C I containing the cytomegalovirus
immediate early (CMVlE) promoter reporting green fluorescent protein
(GFP) to allow repeated observation of Schwann cells either in
monoculture or coculture systems. Following subculture onto monolayers
of embryonic rat dorsal root ganglion neurons under growtharrestdmyelin-forming conditions (Human Placental Serumsupplemented medium preconditioned against growth-arrested cultures),
SCL 4.IF7 cells rapidly ensheathed and myelinated nerve axons. The
constitutively active promoter was used to determine the response time of
the reporter gene and the duration of persistence of its expression in
coculture systems enabling myelin-fomtion. We also investigated the
expression of a neural cell type-specific promoter sequence, the distal
portion of the human neurofilament Lprotein (hNF-L) promoter [3],
which becomes active in Schwann cells which are induced to demyelinate
[4] but is suppressed in myelinatingSchwann cells. The NF-L promoter
sequence was inserted between BamHl and Psrl sites of plasmid pEGFP1 containing the GFP coding region. The plasmid (pEGFP-NFL) was
transfected into SCU. 1/F7 cells as before.
Cells transfected with pEGF-CI expressed GFP during
proliferation (Fig 1A) and after growth arrest. Cells also expressed GFP
when transfected during or after growth-arrest (Fig. IB). When under
control of the distal hNF-L promoter sequence, constitutive activity was
seen in SCIA.lF7 cells adopting process-bearing morphology during
early differentiation [2], however when transfected cells were allowed to
differentiate further, expression was reduced in some cells and was
absent in others (Fig 1C.D). pEGF-CI expression in SCL4.1/F7 was
stable following passage. and transfer to coculture and GFP expression
could be visualized repetitively in single cells contacting axons up to two
weeks after transfection (Fig lE,F).
An alternative transduction method was developed using
replicationdefective adenovirus (RAd35). GFP or P-galactosidase
structural genes were inserted into the Nhel site of plasmid pAL119,
which provides adenovirus flanking sequences. Recombined adenoviruses
are generated following co-tnlnsfection of the transfer vector with the
plasmid pJM17 using methodology developed previously as described
[5]. The adenovims flanking regions allow homologous recombination
with the adenovirus genome provided in PJM17, enabling the insertion of
either GFP or P-galactosidase into the recombinant virus Reporter gene
expression by this method was near universal in cultures of growing
SCU.I/F7 cells, enabling population of single axons in coculture by
several myelin-formingcells expressing reporter constructs (Fig IG).
These preliminary studies show that clonally-derived Schwann
cells can exhibit sustained expression of reporter genes, under the control
of both constitutive and eukaryotic neural cell-specific promoters, which
persists during nerve axon ensheathment in virro and does not alter
n o d patterns of cell differentiation. This model should provide more
detailed information on the way gene expression is controlled by neuronglial interaction during myelinatiotddemyelination of peripheral nerves.
Figure 1
Expression of reporter constructs in SCLA.l/F7 cells in monoculture and
in coculture with embryonic sensory neurons
A.B. GFP expression in growing cells transfected with plasmid pEGF-Cl
24h (A) and 72h (B) after plating and observed 24 h after transfection.
C,D. GFP expression in cells transfected with pEGFP-NFL 24h after
plating and allowed to differentiate spontaneously to process-bearing (C)
and tenninallydifferentiated (D) morphology. Cells which are
completely differentiated ( m w e d ) have ceased expression of the
reporter. E. Myelin-formation in cocultutes (arrowed). F. SCL4.1IF7 cell
expressing GFP in the same field. G. Population of an axon in coculture
by multiple SCL4.UF7 cells expressing a CMVIW~galactosidase
reporter construct using RAd35 as vector. MagnificationXIZS.
We acknowledge support of the Multiple Sclerosis Action Committee and
Surrey Action for Multiple Sclerosis (SAMS). S.S. was supported by a
CAMR short-term studentship.
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