Proc. Natl Acad. Sci. USA Vol. 78, No. 7, pp. 4383-4387, July 1981 Cell

Biological activity of cloned retroviral DNA in microinjected cells (long terminal repeat/transcription/promoter assay) JOHN J. KOPCHICK, GRACE Ju, A. M. SKALKA, AND DENNIS W. STACEY Roche Institute of , Nutley, NewJersey 07110 Communicated by B. L. Horecker, April 1, 1981

ABSTRACT Avian retroviral DNA molecules that had been 2.2) (2). The LTR is believed to contain the promoter se- cloned from infected cells by using recombinant DNA techniques quence(s) for viral transcription (8, 9). were microinjected into either uninfected chicken embryo fibro- Because the enzyme Sal I used to clone the circular DNA blasts (CEF) or CEF transformed by the envelope glycoprotein- cleaves within the viral env gene (1, 2, 9), the cloned molecules deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Ret- contained a permuted gene order with portions of env gene at roviral DNA injected into RSV(-) cells directed transcription of each terminus of the virus-specific region within the linear A envelope mRNA, which was then able to complement the RSV(-) vector (Fig. IA) (2). Therefore, the production of env mRNA env deficiency and promote the production of infectious trans- in a recipient cell would require (i) removal of virus-specific forming virus. The retroviral DNA also directed the production DNA from the A vector, (ii) ligation of the ends of the cloned of fully infectious virus after injection into uninfected cells or and RSV(-) cells. Virus production began within 3-4 hr after microin- DNA to form a circle or linear concatemer (Fig. 1B), (iii) jections. When 100 DNA molecules per cell were injected, almost transcription of the injected DNA. Subsequent translation of all injected cells produced infectious virus. As the number of in- the mRNA would produce the envelope precursor protein, jected molecules per cell was decreased, a corresponding decrease which would be processed and incorporated into budding was observed in the number ofcells that produced infectious virus. virions. DNA injected into the cytoplasm was 1/50th to 1/10th as effective The result reported here suggests that DNA ligation and in virus production as DNA molecules injected into the nucleus. transcription occur rapidly and directly from injected DNA DNA molecules containing one or two tandem copies of the viral molecules. The data also indicate that our microinjection system long terminal repeat were equally effective in virus production. provides a sensitive assay in which to explore the relationship between eukaryotic promoter structure and function. DNA , restriction endonuclease mapping, and nucleo- tide sequence determination experiments have revealed many MATERIALS AND METHODS details of the molecular structure of avian retroviruses (1-10). Cell Culture. CEF preparations and culture conditions have In order to correlate structural datawith information concerning been described (14-16). CEF preparations were negative in the function, we have studied the biological activity ofcloned retro- expression of endogenous group-specific antigens and endog- viral DNA molecules after microinjection into cultured chicken enous env activity (gs-, chf; from SPAFAS). RSV(-) refers to cells. CEF infected with the Bryan strain of RSV in the presence of In previous reports, viral RNA molecules were studied by ultraviolet-light-inactivated Sendai virus (14-16). RSV(-) vi- using the technique of microinjection with glass micropipettes rions produced by RSV(-)-transformed cells contain no helper (11-13). For these studies, injections were performed into virus (11). No virus infectious for CEF (C/E type, which are chicken embryo fibroblasts (CEF) transformed by the envelope resistant to infection with subgroup E virus) were released from glycoprotein-deficient Bryan strain of Rous sarcoma virus RSV(-)-transformed cells. [RSV(-) cells]. Envelope glycoprotein formed within the in- Microinjection. For each microinjection experiment, RSV(-) jected cells complemented the env deficiency ofthe Bryan RSV, cells from the same preparation were plated onto individual resulting in the production of focus-forming units (FFU) of in- glass coverslips. Prior to injection, the coverslip was placed in fectious RSV. With this sensitive assay env mRNA and its nu- a 35-mm plate and incubated with 2 ml of growth medium at clear precursor were detected in cells and virus particles. FFU 370C for 1 hr. Microinjection using glass pipettes with an out- production began approximately 3 hr after RNA injection and side diameter of 0.5 um was performed on 300 cells at a mag- continued for 48 hr. A direct relationship was observed between nification of X640 in approximately 30 min at room tempera- FFU production and the amount of env mRNA injected. ture. A glass syringe was used manually to produce and regulate In the studies presented in this paper, cloned retroviral pressure necessary for microinjections. We also have performed were injected into RSV(-) cells as well as normal un- microinjections with uninterrupted flow from the micropipette infected CEF. Transcription of the injected DNA was assayed and found no difference in results between cytoplasmic and by the production ofinfectious virus. The cloned DNA used in nuclear injections. This pressure was created only when the orifice of the micropipette was within the cell. Cytoplasmic in- these studies originated from transformation-defective uninte- jections were visualized by movements of granules away from grated circular molecules isolated from CEF infected by the site of the cytoplasm at the site ofinjection. Nuclear injec- Schmidt-Ruppin B RSV (td SR-RSV-B). This DNA, cloned in tions were apparent with an immediate change ofthe refractive the bacteriophage A vector Charon 21A, was shown previously index of the nucleoplasm upon microinjection (17). Spillage of to contain either one (ASRBtd-2.4) or a mixture ofone and two tandem copies ofthe viral long terminal repeat (LTR) (ASRBtd- Abbreviations: CEF, chicken embryo fibroblasts; RSV, Rous sarcoma virus; RSV(-) cells, CEF transformed by envelope glycoprotein-defi- The publication costs ofthis article were defrayed in part by page charge cient RSV; SR-RSV-B, Schmidt-Ruppin strain B of RSV; td, transfor- payment. This article must therefore be hereby marked "advertise- mation-defective; FFU, focus-forming units; LTR, long terminal ment" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact. repeat. 4383 Downloaded by guest on October 3, 2021 4384 Cell Biology: Kopchick et al. Proc. Natl. Acad. Sci. USA 78 (1981)

Sal I Sol I 4 LTR 4 A I C F a gog pozzzi

Sal I Sal I Sal I *I LTR * LTR 4 B NZA c gag pol c/// gag pol

FIG. 1. (A) Sal I restriction endonuclease map of the ASRBtd-2.4 recombinant clone. The env gene is indicated by hatching and the LTR by stippling. Sal I sites indicate the boundary between bacteriophage A DNA and virus-specific DNA. The relative order ofthe viral genes in the viral RNA genome is: (5')gag,pol, env, C(3'). The virus-specific sequence in this clone is permuted sa as to divide the env gene. (B) Diagram ofa concatemer of virus-specific DNA molecules. Virus-specific DNA (shown in A) was freed from bacteriophage A sequences by Sal I treatment prior to microin- jection. If two (or more) such molecules were ligated together after injection, the DNA molecule diagrammed above would result. This molecule contains a structure, noted by broken lines, analogous to proviral DNA integrated in the chromosome of an infected cell. It is conceivable that this structure would be transcribed as though it were a normal provirus.

solutions into the cytoplasm during nuclear injections could not Recombinant DNA was handled in accordance with the be completely avoided. After injections, the coverslip was guidelines of the National Institutes of Health. placed in 2 ml of growth medium and incubated at 370C for various time intervals. After incubation, the entire 2 ml ofcul- RESULTS ture fluid was collected for virus assay. Assays for env mRNA Production: Time Course of RSV Infectious RSV production by injected RSV(-) cells was de- Release. Virus-specific DNA was freed from the A vector DNA termined in an infectivity assay for FFU as described (11-13). by cleavage with Sal I and purified by sucrose gradient sedi- Infectious, nontransforming virus (td SR-RSV-B) produced by mentation (see Materials and Methods). When this virus-spe- microinjected normal CEF were assayed indirectly by using the cific DNA was microinjected into the nuclei of RSV(-) trans- following protocol: Culture fluids, collected at various time in- formed cells, infectious RSV were first detected at 3 hr after tervals from microinjected CEF, were added to a culture dish injection, and the number increased continuously for 48 hr containing 2.0 X 105 CEF and 1 X 105 RSV(-)-transformed thereafter (Table 1A). This is similar to the time course ofvirus cells,,followed by incubation at 370C for 48 hr. Infectious RSV production observed after injections of env mRNA into the cy- produced by these infected cultures was determined as de- toplasm ofRSV(-) cells (11). When virus-specific DNA was in- scribed above (11-13). jected into the cytoplasm ofRSV(-) cells, virus production also The following values were used in the calculation ofthe num- began at 3 hr (Table 1). Thus, it appears that the transport of bers of DNA molecules microinjected per cell: (i) the final con- the injected molecules into the nucleus occurred rapidly. The centration of cloned DNA used in a given microinjection ex- results presented in Table 1 indicate that nuclear injections are periment (0.2-200 ,ug/ml); (ii) the total volume microinjected about 4- to 6-fold more efficient in the production ofinfectious per cell, which has been determined to be 5.0 x 10-11 ml for virus from RSV(-) cells relative to cytoplasmic injections. Be- cytoplasmic injections and approximately 2.0 x 10-11 ml for cause the volume of material injected into the cytoplasm is 3- nuclear injections (17); and (iii) an average molecular weight for to 4-fold greater than that of nuclear injections, the total dif- the cloned viral DNA of 5.1 x 106 (2). ference in efficiency is approximately 10- to 25-fold. We have Molecular Cloning of SR-RSV-B. The SR-RSV-B td viral repeated these experiments and, in general, find nuclear in- DNA used in these experiments was derived as described (16). jections to be approximately 10- to 50-fold more efficient rela- Molecular cloning of the td component of unintegrated viral tive to cytoplasmic injections (data not shown). DNA digested with the restriction enzyme Sal I [which was In order to investigate the biological role ofthe LTR in retro- found to cleave the circular molecules at a single location within viral DNA, molecules with either one (ASRBtd-2.4) or a mixture the env gene (1, 2, 9)] into the A vector Charon 21A was as de- of one and two tandem LTRs (ASRBtd-2.2) were injected into scribed (ref. 2; Fig. 1A). In these experiments we used DNA the nucleus and cytoplasm ofRSV(-) cells. No differences were derived from clones (ASRBtd-2.4 or ASRBtd-2.2) that possess apparent in the two preparations (Table 1A). This result suggests a viral insert of approximately 5.0 x 106 daltons, and contains that one copy of the LTR is sufficient for the activity that we one or two tandem copies, respectively, of the LTR. measure. However, it is possible that concatemers of DNA can DNA was extracted from purified recombinant phages as be formed within injected cells. Such concatemers would con- described (2). After the final phenol extraction, 200 ,ug of pu- tain viral structural genes positioned between copies ofthe LTR rified DNA was dialyzed against 10 mM Tris, pH 7.6/10 mM in an arrangement similar to that of integrated proviral DNA. NaCl/1 mM EDTA and digested with Sal I, and the virus-spe- The formation of such a structure might eliminate the require- cific insert was isolated by sucrose gradient sedimentation. ment for two copies in a single molecule (Fig. 1B). Fractions of the gradient containing the retrovirus-specific in- When the entire A vector DNA molecule containing virus- sert were detected by agarose electrophoresis followed by trans- specific DNA with a permuted gene order (ASRBtd-2.4) (Fig. fer to nitrocellulose filters and hybridization as described (18, 1) was injected into the cytoplasm of RSV(-) cells, no virus 19). Sucrose gradient fractions were pooled, and the DNA was were produced in the injected cultures. This suggests that the precipitated with ethanol and dissolved in sterile 1% KCl before env gene could not be reconstituted by ligation. microinjection. Preparations of ASRBtd-2.2 inserts were com- Assays for Virus Production: Injection of Retroviral DNA posed of DNA molecules containing either one or two tandem into CEF. The studies above tested the ability ofviral DNA to- LTRs in approximately equal numbers as described (2). direct the synthesis of active env mRNA to complement the Downloaded by guest on October 3, 2021 Cell Biology: Kopchick et al. Proc. Natl. Acad. Sci. USA 78 (1981) 4385

Table 1. Time course of release of infectious virus after cytoplasmic or nuclear microinjection ofcloned viral DNA into RSV(-) transformed cells or CEF* A. Assays for env mRNA production, FFU from RSV(-) transformed cells B. Assays for virus production, FFU from CEF Time after ASRBtd-2.4 ASRBtd-2.2 ASRBtd-2.4 ASRBtd-2.2 injection, hr Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm Nucleus 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 3 2 8 0 4 0 0 + + 4 4 12 2 8 + + + + 5 20 30 4 20 + + + + 6 26 62 10 52 + + + + 8 28 96 24 84 + + + + 13 44 159 42 130 + + + + 21 120 >400 64 >400 + + + + 28 >400 >400 304 >400 + + + + 48 >400 >400 >400 >400 + + + + (A) Three hundred nuclear orcytoplasmic injections (100 moleculesper cell) were performed in approximately 30 min atroom temperature. Culture fluids were collected at the indicated time intervals and analyzed for the ability ofinjected DNA to complement the env deficiency ofRSV(-) cells. The numbers indicate the titer of FFU after RSV(-) injections. (B) The presence oftd SR-RSV-B after 300 nuclear or cytoplasmic CEF injections was determined as follows: Culture fluids were collected from the microinjected CEF at the indicated times and transferred to a culture containing CEF and RSV(-) cells as described in Materials and Methods. Released td SR-RSV-B from the microinjected CEF would infect neighboring RSV(-) cells, leading to release oftransforming virus. After incubation for 48 hr, the culture fluids were collected and assayed for transforming virus (FFU). Release of td SR-RSV-B from the original microinjected CEF is indicated by a +. * The data are representative of data collected in a total offour experiments.

RSV(-) genetic deficiency. It was of interest to determine cells (data not shown) and CEF (Table 3). Thus, we conclude whether the injected DNA could also function to direct the syn- that nearly every cell receiving 100 molecules produced infec- thesis of other viral molecules (gag and pol mRNA and viral tious virus. When 10 DNA molecules were injected into each genomic molecules) required in virus production. For this anal- of 300 CEF, those subcultures receiving as little as 20% of the ysis, purified retrovirus-specific DNA (ASRBtd-2.2 or ASRBtd- injected cells went on to produce virus in each of two experi- 2.4) was injected into the nucleus or cytoplasm of uninfected ments. When an average of 1 DNA molecule was injected into CEF. After injections, culture fluids were collected at various the cytoplasm of each of 300 CEF or RSV(-) cells, however, times and assayed for infectious, nontransforming virus (using in four separate experiments, only two of the subcultures re- the helper assay described in Materials and Methods). Infec- leased infectious virus (Table 3). This low level ofactivity might tious virus were detected as early as 3 and 4 hr after each ofthese indicate that only a few of the injected DNA molecules pos- injections (Table 1B) and continued to 48 hr. A similar result sessed the required structure for virus expression. Alterna- was obtained when supernatant fluids from injected RSV(-) tively, some injected molecules might have been inactivated in cells were assayed for the presence of replication-competent the cell, or more than one molecule may be necessary for bi- virus (data not shown). Thus, the injected DNA directed the ological activity. synthesis ofall essential retroviral functions within 3-4 hr after Microinjection ofX-Irradiated Cells. X-ray treatment ofcells injection. No reproducible differences were detected between is known to block retroviral infection (20), although it is not cultures injected with ASRBtd-2.2 or ASRBtd-2.4 DNAs. known at which step in the viral replication cycle the inhibition Effects of DNA Concentration. It was of interest to deter- occurs. In order to determine the stability of injected DNA, mine the number ofDNA molecules per cell necessary to detect injections were performed into x-irradiated cells, in which virus env mRNA activity. Accordingly, approximately 1000, 100, 10, amplification by reinfection could not occur. Thus the only or 1 molecule or purified virus-specific DNA was injected into each of a total of300 RSV(-) cells. Virus production with 1000 Table 2. Virus release after injection of or 100 molecules per injected cell began soon after injection various concentrations of (Table 2). Cultures receiving injections averaging 10 and 1 mol- viral DNA into RSV(-) transformed cells ecules per cell started releasing virus at 11 and 23 hr, respec- FFU released at each number of DNA tively. We presume that the delay in virus production in these Time after molecules per cell injected cultures resulted from the requirement for virus spread and injection, hr 1000 100 10 1 1* amplification in the number ofvirus-producing cells to readily 2-6 8 2 0 0 0 detectable levels. 7-11 44 42 4 0 0 To determine the number of virus-specific DNA molecules 19-23 162 134 38 2 0 required to produce fully competent virus particles, end-point Three hundred cytoplasmic injections ofviral DNA (ASRBtd-2.2) at dilution studies were undertaken. 1000, 100, 10, or 1 DNA the four indicated average concentrations were made into RSV(-) (ASRBtd-2.2) molecule was injected into each of300 CEF, after cells. The ability ofthe injected DNA to complement the env deficiency which the injected culture was subcultured in various dilutions of RSV(-) cells was assayed by determining the number of FFU re- with uninjected cells. When 1000 or 100 molecules per cell were leased by RSV(-) cells at various times after injection. injected all subcultures, even those receiving only 2% of the * Similar or identical results were obtained when ASRBtd-2.4 DNA (containing only one copy ofthe LTR) was injected except in the case injected cells (=6 cells), went on to produce infectious virus. of1 molecule per cell. The last column presents the results from these This was the case in two separate experiments with both RSV(-) injections. Downloaded by guest on October 3, 2021 4386 Cell Biology: Kopchick et al. Proc. Natl. Acad. Sci. USA 78 (1981) Table 3. Virus release after injection ofvarious concentrations of CEF. We have found that this linear DNA could be utilized in viral DNA into CEF the biosynthesis of infectious virus when injected into x-irra- diated CEF (data not shown). % of Production of infectious virus at injected each number ofDNA molecules DISCUSSION cells per cell injected transferred 1000 100 10 1 1* We have analyzed the expression ofcloned DNA after microin- jection into cultured eukaryotic cells. The datademonstrate that 50 + + cloned avian retroviral DNA can be transcribed after microin- 30 + + jection into RSV(-) cells to produce envelope glycoprotein 13 + + mRNA. The injected DNA also directed the synthesis of infec- 5 + + tious virus particles after injection ofuninfected CEF. Because 2 + + 80 + - - the recombinant DNA contained a permuted gene order (Fig. 20 + - + LA), injected molecules had to be ligated together within the cell for env gene expression to take place. As a control the entire Todetermine the numberofindividual CEFable torelease infectious A recombinant DNA molecule containing the bacteriophage virus, various percentages of cultures injected with ASRBtd-2.2 DNA arms, which prevented env gene ligation and reconstitution, were subcultured with uninjected RSV(-) cells 1 hr after injection. td was shown to be unable to direct the synthesis of env mRNA SR-RSV-B releasedby the microinjected CEFwouldinfectneighboring RSV(-) cells, with subsequent release of transforming virus. After or virus particles. culture for 4 days the appearance oftransforming virus or FFU (shown Virus production was first detected approximately 3 hr after bya +) indicates thatatleastonemicroinjected CEFwhen subcultured injection of the virus-specific DNA into the nucleus or cyto- with uninjected RSV(-) cells had released nondefective virus. plasm of RSV(-) cells. A similar delay was observed when env * Results with injection of ASRBtd-2.4 DNA. See footnote for Table 2. mRNA itself was injected into RSV(-) cells (11). This suggests that the steps involved in mRNA production-i.e., ligation of source ofviral DNA in each cell would be that which had been the DNA, transport into the nucleus, transcription, and RNA physically injected, and any env mRNA present within the cul- splicing-required a nearly insignificant amount of time com- ture should be transcribed from that DNA. pared to the approximately 3 hr required for translation of the RSV(-) cells that received 3000 or 10,000 roentgens (1 roent- mRNA, glycosylation of the protein, and its association with gen = 2.6 x 10-4 coulomb/kg) of x-irradiation, along with virus particles. Because transcription occurred so rapidly, it unirradiated cultures, were injected with virus-specific DNA. seems possible that integration of the DNA into a host chro- Parallel cultures were infected with approximately two infec- mosome, as is normally essential for viral RNA synthesis (21), tious Rous-associated virus 2 (RAV-2) particles per cell to de- may not have been required in these experiments. However, termine the effectiveness of irradiation. After cytoplasmic in- it cannot be excluded that one or a few of the many injected jection of virus-specific DNA (100 molecules per cell into 300 molecules had been immediately integrated into host DNA. If cells), virus production from.x-irradiated cells receiving 10,000 a virus-encoded protein is involved in viral DNA integration roentgens was similar to that of untreated cultures. Virus pro- (22, 23) our finding of similar kinetics of expression in RSV(-) duction began at 3 hr, increased until 42 hr, and began to de- and uninfected cells suggests that integration is not required crease after 66 hr, presumably due to cell death caused by ir- for mRNA expression from microinjected DNA. radiation. Virus production continued for at least 120 hr (Table In these experiments molecules containing one copy of the 4). Control experiments confirmed that virus infection had been LTR per genome equivalent were as competent as those con- blocked by the x-ray treatment. taining two. Thus, the reaction we measure does not require These dataindicate not only that the injected DNAwas stable the tandem duplication ofLTRs present in those molecules con- for at least 120 hr but that the block of infection due to x-irra- taining two such structures. Ligation of concatemers of mole- diation occurs before reverse transcription and synthesis ofviral cules containing one LTR would generate a genome flanked by DNA. In this regard, we have investigated the ability of linear LTRs similar to that of integrated proviral DNA (Fig. 1B) (2, unintegrated viral DNA isolated from infected cells to direct the 5, 6). Thus, concatemer formation might eliminate the need for synthesis of infectious virus after injection into x-irradiated two copies of the LTR. Injection of circular molecules, con- Table 4. Microinjection of viral DNA into x-irradiated RSV(-) transformed cells Time after FFU released injection Injected* Infectedt or infection, 3000 10,000 No 3000 10,000 No hr roentgens roentgens treatment roentgens roentgens treatment 3 8 8 6 ND ND ND 9 78 56 100 ND ND ND 18 128 100 160 0 0 54 42 >400 80 >400 66 0 >400 66 >400 220 >400 96 0 >400 90 >400 130 >400 190 0 >400 120 >400 48 >400 288 0 >400 * Three hundred injections of approximately 100 virus-specific DNA molecules (ASRBtd-2.4) each were performed into three RSV(-) cultures. One culture had received no treatment whereas the others had received 3000 or 10,000 roentgens of x-irradiation prior to injection. Culture fluids were collected at various times and analyzed for FFU. t X-irradiated and control unirradiated RSV(-) cultures were infected with approximately two Rous-as- sociated virus 2 particles per cell to determine if virus infection was blocked. ND, not determined. 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taining one or two LTR's with reduced possibility for concate- ified before expressing its biological properties. For example, mer formation, should help to revolve this question. input DNA might be chemically modified or integrated behind When approximately 100 molecules of virus-specific DNA an active cellular DNA sequence-it might be rearranged or were injected into the cytoplasm of RSV(-) cells, a maximal undergo recombination. Further work is required to determine response in transcription of env mRNA was observed soon af- whether any such modifications occur after microinjection. ter injection, and essentially all injected cells were able eventu- However, the fact that transcription can be detected almost im- ally to release infectious virus. When an average of 1 molecule mediately after injection makes it seem likely that it is the input was injected per cell, however, only approximately 1 in 600 DNA, without major alterations, that directs RNA synthesis, cells went on to release virus. This difference in efficiency of and that the viral DNA carries its own promoter for transcription. transcription may reflect one or more of the following circum- stances: First, nonspecific nuclease degradation may reduce the We thank B. Cullen and F. Hishinuma for helpful comments. J.J. K. effective concentration of injected viral DNA. Second, tran- was supported by Postdoctoral Fellowship PF-1793 from the American scription of a complete env mRNA may require linear conca- Cancer Society. temers containing two LTRs. In cells receiving an average of only 1 molecule, concatemer formation could occur only infre- 1. Czernilofsky, A. P., Levinson, A. O., Varmus, H. E., Bishop, J. M., Tischer, E. & Goodman, H. M. (1980) Nature (London) 287, quently in the few cells receiving more than 1 molecule. Third, 198-203. in cells receiving few molecules a less efficient mechanism, such 2. Ju, G., Boone, L. & Skalka, A. M. (1980)J. Virol. 33, 1026-1033. as circularization or integration of the injected molecule, or 3. Skalka, A., DeBona, P., Hishinuma, F. & McClements, W. both, may be required for virus production. (1980) Cold Spring Harbor Symp. Quant. Biol. 44, 1097-1103. Injected DNA molecules were continuously expressed for at 4. DeLorbe, W. J., Luciw, P. A., Goodman, H. M., Varmus, H. E. least 120 hr even in RSV(-) cells x-irradiated prior to injection. & Bishop, J. M. (1980) J. Virol. 36, 50-61. 5. Hsu, W., Sabran, J. L., Mark, G. E., Guntaka, R. V. & Taylor, Because no virus infection could take place within these cul- J. M. (1978) J. Virol. 28, 810-818. tures, only the injected cells were able to release infectious virus 6. Shank, P. R., Hughes, S. H., Kung, H. J., Majors, J. E., Quin- and only the injected DNA (or DNA synthesized by the cell trell, N., Guntaka, R. J., Bishop, J. M. & Varmus, H. E. (1978) using injected DNA as templates) could direct the synthesis of Cell 15, 1383-1395. viral RNA. Inspection of the irradiated culture indicated that 7. Hughes, S. H., Shank, P. R., Spector, D. H., Kung, H. J., the decline in virus production at 120 hr after 10,000 roentgens Bishop, J. M., Varmus, H. E., Vogt, P. K. & Breitman, M. L. (1978) Cell 15, 1397-1410. of x-ray treatment was probably the result of cell death rather 8. Yamamato, T., Jay, G. & Pastan, I. (1980) Proc. Natl. Acad. Sci. than loss of active DNA. USA 77, 176-180. It has been reported that direct microinjection ofthe herpes 9. Ju, G. & Skalka, A. M. (1980) Cell 22, 379-386. simplex virus thymidine kinase gene into the nucleus of cul- 10. Hishinuma, F., DeBona, P., Astrin, S. & Skalka, A. (1981) Cell tured mouse cells deficient in thymidine kinase activity resulted 23, 155-164. in the ability of 50-100% of the cells to express this enzymatic 11. Stacey, D. W., Allfrey, V. G. & Hanafusa, H. (1977) Proc. Natl. Acad. Sci. USA 74, 1614-1618. activity. However, no thymidine kinase activity could be de- 12. Stacey, D. W. & Hanafusa, H. (1978) Nature (London) 273, tected when the DNA was injected into the cytoplasm of the 779-782. cultured mouse cells (24). In contrast to these results, our stud- 13. Stacey, D. W. (1980) Cell 21, 811-820. ies reveal that cytoplasmic injections of cloned retroviral DNA 14. Rubin, H. (1960) Proc. Natl. Acad. Sci. USA 46, 1105-1119. into RSV(-) cells or CEF are 1/50th to 1/10th as efficient rel- 15. Hanafusa, H. (1969) Proc. Natl. Acad. Sci. USA 63, 318-325. ative to nuclear injections. These differences may reflect in- 16. Kawai, S. & Hanafusa, H. (1972) Virology 49, 37-44. 17. Stacey, D. W. & Allfrey, V. G. (1976) Cell 9, 725-732. herent differences in the type of cells injected, properties of 18. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. retroviral DNA, or sensitivities of assays employed. 19. McClements, W., Hanafusa, H., Tilghman, S. & Skalka, A. One of the most interesting findings from this work was the (1978) Proc. Natl. Acad. Sci. USA 76, 2165-2169. rapidity with which the injected DNA can be transcribed after 20. Hanafusa, H., Hanafusa, T. & Rubin, H. (1963) Proc. Natl. Acad. injection. This rapidity together with the extreme sensitivity Sci. USA 49, 572-580. 21. Bishop, J. M. (1978) Annu. Rev. Biochem. 47, 35-88. exhibited by our biological assay system can provide an impor- 22. Schiff, R. D. & Grandgenett, D. P. (1978)J. Virol. 28, 279-291. tant system to test for DNA function. One concern regarding 23. Kopchick, J. J., Harless, J., Geisser, B. S., Killham, R., Hewitt, assays performed within viable cells such as microinjection or R. R. & Arlinghaus, R. B. (1980) J. Virol. 37, 274-283. DNA is the possibility that the sample will be mod- 24. Capecchi, M. R. (1980) Cell 22, 479488. Downloaded by guest on October 3, 2021