Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or beta-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa, 293, or NB324K cells, in increasing order of efficiency. When homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (-) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired.

We previously reported that a recombinant genome derived from the autonomous rodent parvovirus Lulll could be pseudotyped with capsids of the closely related viruses, H 1 and minute virus of mice. To determine whether this was also possible with less related viruses, Lulll recombinant genomes containing a luciferase reporter were cotransfected into permissive cells together with plasmids expressing the capsid proteins of either feline panleukopenia virus (FPV) or its host range variant, canine parvovirus (CPV). We observed efficient packaging of the recombinant DNA into transducing virions that displayed the cell tropism of the virus that supplied the capsid. Thus, the FPV-and CPVpseudotyped virions were able to transduce a feline cell line but they showed no transducing activity for the human NB324K line, which is permissive for Lulll. The transducing activity of the pseudotyped viruses was not inhibited by neuraminidase treatment of the permissive recipient cells, in contrast to that of virions packaged using Lulll capsid proteins. Furthermore, canine A72 cells (permissive for CPV but not FPV) were efficiently transduced by CPVpackaged but not by FPV-packaged Lulll recombinant genomes. Pseudotyped recombinants will be useful for elucidating parvovirus host range determinants since they enable the packaged DNA and each of the capsid proteins to be supplied independently. They should also facilitate control over the targeting of parvovirus vectors for gene transfer.

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Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K), we found that the serum-free alternative preserves nearly as efficiently as the serumcontaining preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

rent pattern. Using a porous agarose layer to support the sample, we frequently measure proton currents traveling towards the dish floor, suggesting that this provides a more physiological environment in which ions are free to move from all surfaces of the ovariole unimpeded (Figure 3).The recording chamber provides a physiological environment and specimen manipulation and orientation possibilities that are well suited to extracellular electrophysiological investigation of a variety of tissues and can be adapted to other studies requiring precise sample orientation. REFERENCE 1. Diehl-Jones, W.L. and E. Huebner. 1992. Spatial and temporal transcellular current patterns during oogenesis. Dev. Biol. 153 :302-311.Canadian NSERC research and equipment grants to E.H. are gratefully acknowledged. We thank C.