Transient transfection of ecotropic retrovirus receptor permits stable gene
transfer into non-rodent cells with murine retroviral vectors
Transient transfection of ecotropic retrovirus receptor permits stable gene transfer into non-rodent cells with murine retroviral vectors
Axel
Scholz
and
Miguel
Beato*
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-Mannkopff Strasse 2, D-35037
Marburg
,
Germany
Received December 11, 1995;
Accepted January 11, 1996
Retroviral vectors are useful for efficient, stable and single copy transfer of
foreign genes into animal cells. However, the low risk murine ecotropic vectors
can only be used with rodent cells due to their restricted host range. When
using cells from other species, in particular human, amphotropic retroviral
vectors and the corresponding packaging cell lines are required. Given the wide
host range of these vectors, they are classified as higher biological risk and
high biological containment (L2 or even L3) is mandatory in many countries. To
reduce the risk to the experimenter while working with non-rodent cells, we considered a strategy based on transient expression of
the recently cloned murine ecotropic retrovirus receptor (
1
) to allow the use of safer murine retroviral vectors.
Murine retroviruses enter target cells by interaction of the viral envelope
glycoprotein with a specific cell-surface receptor, a basic amino acid transporter (
2
,
3
). The retroviral host range is determined by the species- and/or tissue-specific expression of the appropriate receptor. To extend the host
range of the rodent-specific Moloney murine leukemia virus we transiently transfected the expression vector for the virus receptor, pJET (
1
), into endometrial epithelial cells from rabbit, RBE7 (
4
), or human, Ishikawa (
5
), via the calcium phosphate precipitate technique. Transfection efficiencies of
5-10% were reached as determined by co-transfection of a RSV-LacZ vector followed by cytochemical staining for [beta]-galactosidase activity. Following removal of the
precipitate, 16 h after transfection, the cells were co-cultivated with the packaging cell line psi2 expressing v-Ha-
ras
and the neo gene as described (
6
). After 3-5 days of exposure to high virus titres (10
6
c.f.u./ml), the cells were selected for 4-6 weeks on G418 and the number of transformed clones was counted. Whereas
in mock transfected cells no foci were detected, a large number of foci was
generated in cells transfected with the murine ecotropic vector prior to
infection. The number of foci obtained with 1 * 10
6
cells was in the range of 400 for RBE7 cells and 50 for Ishikawa cells. These
transformation efficiencies are within the range obtained with a rat cell line
of endometrial origin, RENT4 (
6
), infected with the same protocol. Southern blot analysis of five independent
RBE7 foci demonstrated single copy retroviral integration (Fig.
1
B). Homogeneous expression of the v-Ha-
ras
oncogene was demonstrated by immunofluorescence with anti Ha-
ras
antibodies, which decorated the cytoplasm only in transformed cells but not in
control cells (Fig.
2
).
REFERENCES
1 Albritton, L.M., Tseng, L., Scadden, D. and Cunningham, J.M. (1989) Cell 57, 659-666.MEDLINE Abstract
2 Wang, H., Kavanaugh, M.P., North, R.A. and Kabat, D. (1991) Nature352, 729-731.MEDLINE Abstract
3 Kim, J.W., Closs, E.I., Albritton, L.M. and Cunningham, J.M. (1991) Nature 352, 725--728.MEDLINE Abstract
