Proc. Nati. Acad. Sci. USA Vol. 84, pp. 2781-2784, May 1987 Developmental Biology Rous sarcoma is integrated but not expressed in early embryonic cells (/early development/gene expression) E. MITRANI*t, J. COFFINS, H. BOEDTKER*, AND P. DOTY* *Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138; and tDepartment of Molecular Biology and Microbiology and Cancer Research Center, Tufts University School of Medicine, Boston, MA 02111 Contributed by P. Doty, December 22, 1986

ABSTRACT We have developed a protocol that allows us grown in ovo and in vitro, and primary cells from various to infect chicken early embryonic (CEE) cells with high stages and from different areas can be grown in culture efficiency. This was achieved by exposing the CEE cells to a (9-12). Salter et al. (13) have reported the stable integration semicontinuous dose of Rous sarcoma virus (RSV) for a period of proviral Rous sarcoma virus (RSV) into chicken embryo of20 hr. Southern blot analysis indicated that an average ofone germ cells after in ovo with RSV. Yet, the mecha- proviral copy is integrated per embryonic cell. However, there nisms of interaction between RSV and the embryonic cells was no production of infectious viral particles by the cells and the stage at which the embryonic cells became infected containing the proviral , although low levels of full- have not been determined. In the present work we present length genomic RNA could be detected by RNA transfer blot data that shows that normal early chicken embryonic cells analysis. These low RNA levels contrast with the 100- to can be infected at high efficiency with RSV. We also show 1000-fold higher levels found in RSV-infected chicken embryo that in spite ofthis, the levels ofviral mRNA produced by the fibroblasts. We conclude that in cells derived from pregastru- infected cells are very low and are not sufficient to generate lating chicken embryos, RSV DNA is integrated into the cell fully competent viral particles. genome but fails to be expressed in an efficient manner. These primary cells can therefore be used to identify factors involved MATERIALS AND METHODS in regulation ofretroviral gene expression in normal cells. Such factors may also be instrumental in elucidating basic mecha- Preparation, Culture, and Retroviral Infection of Chicken nisms involved in gene regulation during early development in Early Embryo (CEE) Cells. Stage X blastoderms were iso- higher vertebrates. lated from commercial unincubated eggs provided by SPAFAS. Most unincubated eggs obtained were found to be RNA tumor (retroviruses) have been studied exten- at stages later than stage X. A special arrangement was sively because of their capacity to induce tumors, their therefore made with SPAFAS to obtain eggs that had been particular replication strategies, and their wide distribution in collected immediately after laying. After dissection, only nature as agents that can be transmitted horizontally and those blastoderms found to be at stages X-XI (14) were used. genetically (1). Retroviruses are now becoming increasingly Whole embryos were then trypsinized as described (12) and useful as vectors for gene transfer into cells (2, 3). This dissociated in culture medium supplemented with RSV at capacity, however, has been severely hampered by the lack 106-107 focus-forming units (ffu)/ml. The cells were plated at ofprovirus expression once introduced into early embryos or a concentration of 1.5 x 105 cells per ml. Twenty-four hours embryonic stem cells (4, 5). In the mouse, preimplantation later the medium was replaced with virus-free medium and embryos are susceptible to integration but cannot the cells were incubated further for various periods of time express viral functions (6). In contrast, in postimplantation (protocol I). When CEE cells were exposed to a semicontinu- mouse embryos, retrovirus can integrate as well as express ous dose of RSV (protocol II), the initial medium was high concentrations ofviral-specific in every organ (7). replaced 12 hr later and then at 4-hr intervals, for the next 12 Attempts at understanding this block of retroviral expression hr, with fresh medium containing RSV at a concentration of in early mouse embryonic cells have led to the proposal of 106 ffu/ml. The strain used, Prague RSV, subgroup B (Pr B generalized mechanisms of gene regulation that may operate RSV), has been described (15). The stock used contained in early development, such as de novo DNA methylation (4). about 50% of mutants lacking part of the src gene. In addition, certain regulatory sequences ofretrovirus do not DNA Analysis. At the end of the culture period, the cells function in embryonic carcinoma (EC) cells unless the were rinsed five times in phosphate-buffered saline (PBS), enhancer sequence is deleted. It has thus been proposed that removed with a rubber policeman, and digested with pro- undifferentiated cells contain trans-acting regulatory factors teinase K (200 ,g/ml) at 45°C for 45 min. The cell digest was that reduce transcription by interacting with viral enhancers then extracted twice with phenol/chloroform. Sodium ace- (8). tate to 0.3 M and 2 vol of ethanol were added to half of the In an effort to examine to what extent what has been material, which was stored and processed later to prepare learned about retroviral behavior in the early mouse embryo RNA. The other half was digested with boiled can be generalized to other early developmental systems we pancreatic have now examined retroviral integration and expression in RNase (120 ,ug/ml) at 37°C for 2.5 hr and dialyzed extensively the pregastrulating early chicken embryo. The advantage of in 10 mM Tris HCl/1 mM EDTA (TE), pH 8.0. The DNA was the early chicken embryo is that it contains about 2 orders of precipitated and resuspended in a small volume of TE, and magnitude more cells at the blastula stage than the corre- sponding mammalian embryo. It can be operated upon and Abbreviations: RSV, Rous sarcoma virus; CEE, chicken early embryonic; EC, embryonic carcinoma; Pr B RSV, Prague RSV, subgroup B; Pr C RSV, Prague RSV, subgroup C; kb, kilobase(s). The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at the present payment. This article must therefore be hereby marked "advertisement" address: Institute of Life Sciences, The Hebrew University of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Jerusalem, Jerusalem 91904, Israel. 2781 2782 Developmental Biology: Mitrani et al. Proc. Natl. Acad. Sci. USA 84 (1987) the concentration was determined by measuring the optical A 2 B 3 4 absorbance at 260 nm. The concentration was used through- out as a reference point so that quantities of RNA could be full _ full calculated as DNA equivalents. DNA was digested with Sac length length I restriction enzyme (Bethesda Research Laboratories). Samples (10 jig per lane) were electrophoresed on a 1% agarose gel, transferred to a nitrocellulose filter, and hybrid- a to ized with 32P-labeled riboprobe complementary bases 103-2870 of the Prague RSV subgroup C (Pr C RSV) genome (a gift from D. Hanahan). 28s RNA Analysis. Total nucleic acids obtained as described above were precipitated, resuspended in 10 mM Tris HCl/10

mM MgCl2, and digested with DNase I (10 ,ug/ml) for 30 min src at 370G. RNA was then denatured and electrophoresed on a 1% agarose/formaldehyde gel, transferred to a GeneScreen 8s0 t.i nylon membrane, and hybridized to a 32P-labeled riboprobe complementary to bases 103-255 in the leader region of the Pr C RSV genome (a gift from S. Herman).

RESULTS Southern blots of DNA isolated from the RSV-treated CEE cells (protocol I) using a riboprobe complementary to bases 103-2870 of the RSV genome could only detect the endoge- C RSV spliced mRNAs nous proviral components. In one experiment, in which the cells were grown for 3 weeks before the DNA was processed, a faint band (not shown) was detected that corresponded to 0 1 2 3 4 5 6 7 8 9 Kb I ,-.I, I I 1 . . <10% of the cells being infected. This suggested that some cells were incorporating the proviral genome after "pulse" gag pei env Srrc administration of RSV but that the infection did not spread -rcaDw~ t- an 9.3 env arc through the culture as would be expected if competent cap m an 4.6 particles that could infect other cells were produced. Addi- arc tional experiments were thus performed in which cells were zsapl ana 2.7 grown in the constant presence of RSV in culture to obtain a FIG. 2. RSV-infected CEE cells produce very low levels of viral higher initial multiplicity of infection (protocol II). Under RNA. RNA, extracted from cultured cells 72 hr after RSV infection, these experimental conditions, RSV does integrate into was denatured and electrophoresed on a 1% agarose/formaldehyde genomic DNA of CEE cells as shown in Fig. 1. Since the gel, transferred to a GeneScreen nylon membrane, and hybridized to intensity of the band corresponding to the newly integrated a 32P-labeled riboprobe complementary to bases 103-255 in the provirus is roughly the same as that ofeach ofthe single-copy leader region of the RSV genome. (A) Lane 1, RNA extracted from endogenous components, this indicates an average of about chicken embryo fibroblasts infected with Pr B RSV (positive con- trol). Lane 2, CEE cells grown at high density and infected with Pr one copy of RSV DNA per CEE cell. B RSV. RNA in lane 2 was extracted from 10 times as many cells as To understand why CEE cells did not transmit competent that in lane 1. (B) Autoradiograph, exposed for a longer period of virus to other cells, RNA, extracted 72 hr after infection, was time, of a transfer blot of RNA extracted from untreated CEE cells analyzed on RNA transfer blots using a riboprobe comple- (lane 3) and from RSV-infected CEE cells (lane 4). Even at these mentary to bases 103-255 in the leader region of the RSV levels of exposure, no bands corresponding to env and src mRNAs genome. Lane 1 of Fig. 2A shows RNA extracted from can be detected. The riboprobe also hybridizes to rRNA. The fibroblasts infected with RSV. Lane 2 shows presence of more than one band in the 9-kb region (arrows) is due to chicken embryo heterogeneity of the viral stock, which contained mutants in the src Diagram of RSV spliced mRNAs. m 1 2 3 region (9). (C) 9.4 CEE cells grown at high density and infected with RSV. VW Although RNA in lane 2 was extracted from 10 times as many 6.7 cells as that in lane 1, the bands that hybridized to the viral probe in this lane are weaker by a factor of -10 than those in lane 1. Thus, RSV-infected CEE cells produce only very 4.4 low levels of RNA (less by a factor of -100 than RSV- infected embryo fibroblasts in this case). In no case did we detect viral RNA levels higher than this in CEE cells infected FiG. 1. RSV is integrated in CEE cell genomic DNA. Southern with RSV. Depending on which cells were used as positive blot of genomic DNA from RSV-infected CEE cells. DNA was controls, the levels in CEE cells were lower by a factor of cleaved with Sac I, fractionated on a 1% agarose gel, transferred to 1000 than those produced by RSV-infected embryo fibro- a nitrocellulose filter, and hybridized with a 32P-labeled riboprobe blasts. Since productively infected chicken embryo fibro- complementary to bases 103-2870 of the RSV genome. Lane m, X blasts contain 1-10,000 copies of virus RNA per cell (3), one phage cut with HindIII as marker. Lane 1, turkey fibroblasts infected can estimate that infected CEE cells contain each about 1-10 with Pr B RSV (positive control) showing the integrated proviral Sac copies of viral RNA. Fig. 2B shows an autoradiograph I fragment of 6.6 kilobases (kb). Lane 2, CEE cells infected with a exposed for a longer period of time from a transfer blot of semicontinuous dose of Pr B RSV over a 24-hr period. Lane 3, uninfected CEE cells. The two high molecular weight bands, also RNA extracted from untreated CEE cells (lane 3) and from seen on the uninfected cells, are derived from the endogenous RSV-infected CEE cells (lane 4). Even at these levels of provirus. Note that turkey fibroblasts do not contain crosshybrid- exposure, no bands corresponding to env and src mRNAs izing endogenous proviruses. were detected in three different experiments in which the Developmental Biology: Mitrani et A Proc. Natl. Acad. Sci. USA 84 (1987) 2783 cells were grown at high density and RNA was extracted after infecting F9 cells with a recombinant retrovirus carrying the 72 hr. In another experiment in which the cells were grown neomycin gene (34-36) and characterizing rare colonies that at high density and RNA was extracted after 8 days, no env grew in the presence of antibiotic G418, they found that some and src mRNA bands were detected either. The intensities of proviruses were expressed as a result of being integrated the full-length viral RNA bands were, however, considerably close to specific cellular genomic sequences that apparently weaker. We did observe, nonetheless, faint (<100-fold) env promoted proviral gene activity. An additional case was and src mRNA bands in one case in which RNA was found in which the provirus was expressed due to a point extracted after 8 days but the CEE cells were grown instead mutation in the tRNA primer binding site of the proviral at low rather than high density. In all cases studied, however, genome, although how this point mutation increases viral the common feature was that CEE cells infected at high expression is not yet clearly understood (33). efficiency with RSV produced only very low levels of RNA. Studies of interactions between retroviruses and normal cells have proved very fruitful, in particular in characterizing DISCUSSION various and growth factors (3, 35, 37, 38). Retro- viruses are also becoming indispensable vectors for intro- The method described here allows one to infect CEE cells ducing foreign genes in a stable manner into various cellular with an efficiency of about one proviral copy per cell. and animal systems (2, 3). Yet, the way in which retrovirus Rubenstein et al. (16) have been able to introduce genes into interact with eukaryotic cells is poorly understood. We have transgenic mice by infecting early embryos with defective shown here that in cells derived from pregastrulating chicken retroviruses. They estimated, however, that only about 20% embryos, RSV retrovirus is integrated into the cell genome of the cells in transgenic fetuses contained the proviral but fails to be expressed in an efficient manner. This indicates genome. Even lower efficiencies of infection have been that such a pattern of behavior, previously found only in the achieved when using retrovirus in other transgenic systems mouse system, seems to be of a more universal nature. (17, 18). Furthermore, the range of viruses that may be affected by Even though the CEE cells were infected at high efficien- such a mechanism seems to be equally broad. Thus, we cy, the levels of viral RNA produced in all cases studied were believe that CEE primary cells may be useful for defining low and were not sufficient to generate fully competent viral factors that may be involved in regulation of retroviral gene particles. In most cases the low levels of RNA consisted of expression. Such factors may also be instrumental in eluci- unspliced genomic RNA. At present we do not know whether dating basic mechanisms involved in gene regulation during the unspliced RNA detected is, at least in part, genomic RNA early development in higher vertebrates. that remained in the culture from the time of infection or newly synthesized unspliced RNA. It is also possible that the This research was supported by Public Health Service Grants HD expression of env and src mRNAs varies with the culture 01229 and HD 20705. E.M. was supported in part by Fogarty conditions. Splicing of viral RNA has been a controversial International Fellowship 1 F05 TW03733-01. issue. It has been reported that simian virus 40 RNA is not spliced in undifferentiated murine teratocarcinoma stem cells 1. Varmus, H. E. (1982) Science 216, 812-820. (19), but those data have been questioned recently by others 2. Mann, R., Mulligan, R. C. & Baltimore, D. (1983) Cell 33, (8). In our experiments, the levels of RNA produced were 153-159. clearly not sufficient to generate competent viral particles. 3. Weiss, R., Teich, N., Varmus, H. & Coffin, J. (1984) RNA The situation described here is similar to what has been Tumor Viruses (Cold Spring Harbor Laboratory, Cold Spring observed in early mouse embryos and in EC cells. Other Harbor, NY), Suppl., pp. 36-64. workers have found that mouse undifferentiated EC cells do 4. Jahner, D., Stuhlmann, H., Steward, C. L., Harbers, K., produce low levels of viral RNA after retroviral infection, Lohler, J., Simon, I. & Jaenisch, R. (1982) Nature (London) in 298, 623-628. even though no viral particles can be detected these cells 5. 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