Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10051-10055, November 1992 Developmental Biology
Antisense c-myc effects on preimplantation mouse embryo development BIBHASH C. PARIA*t, SUDHANSU K. DEY*t, AND GLEN K. ANDREWSt§ Departments of *Obstetrics-Gynecology, tPhysiology, and tBiochemistry and Molecular Biology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, KS 66103
Communicated by Clement L. Markert, July 6, 1992
ABSTRACT Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mehanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this
DNA-binding protein could be important during early embryogenesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containg an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eightcell/morula stage. At lower concentrations (7.5 FsM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 FM), all unmdified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 pM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with hih concentrations of the modified sense complement (30 FM). Culture of freshly recovered eight-cell embryos with antisene c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc fmction becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development.
The fertilized mammalian egg, after completion of several cleavages, differentiates into a blastocyst. The blastocyst is composed of the inner cell mass, which gives rise to the embryo proper and certain extraembryonic membranes, and the trophectoderm, which participates in the formation of extraembryonic structures (placenta and parietal yolk sac). Although activation of transcription of the embryonic genome occurs at the two-cell stage in the mouse (1, 2), our knowledge of genes that are crucial for preimplantation embryo development is meager. In this study, the role of c-myc in preimplantation mouse embryo development was examined. The c-myc protooncogene is the cellular homolog of the viral v-myc oncogene and is a member of a gene family that includes L-myc and N-myc (3-7). c-myc is a nuclear DNA-binding phosphoprotein that is highly conserved The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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throughout evolution. It is now thought that biological functions and sequence-specific DNA-binding activity of c-myc are executed by dimerization with another protein called Max (8-10). The expression of c-myc is positively correlated with DNA synthesis and cell proliferation (8-10) and is rapidly induced in various cell types after stimulation with mitogen (11). A direct role of c-myc in the cell cycle is suggested by the observation that quiescent cells enter the cell cycle and divide after microinjection of c-myc protein or transfection with the c-myc gene (12, 13). Because of the association of c-myc with cell cycle in cultured cells (14, 15) and the suggested role of this protein in cell division and differentiation during postimplantation mammalian development (1619), this protein could be important for cleavage and/or differentiation during early mammalian embryogenesis. Definitive proof for a role of c-myc in preimplantation embryo development requires examination of the effects of selective ablation of expression of this gene. Because of the extremely short half-life of c-myc mRNA and protein (20-22), the use of an antisense oligodeoxyribonucleotide (oligo) to c-myc mRNA to block its translation appeared an attractive approach toward elucidating the role ofthis protooncogene in preimplantation embryo development. Since the discovery of antisense RNAs as natural suppressors of prokaryotic genes (23-27), the successful use of antisense RNA/DNA to manipulate expression of endogenous and exogenous eukaryotic genes has been reported (28-34). Furthermore, several laboratories have reported inhibition of cell proliferation resulting from antisense blockage of expression of c-myc during culture in medium containing antisense oligo (35-38). MATERIALS AND METHODS Materials. HPLC-purified modified and unmodified sense and antisense oligos were purchased from Genosys, Houston. Rabbit polyclonal antibodies to human c-myc were kindly provided by Rosemary Watt (Smith Kline & French). Crystalline bovine serum albumin was purchased from Sigma (A-4378). DNA ladder was obtained from Bethesda Research Laboratories. All other reagents were purchased from Sigma, Fisher, or Mallinckrodt. Animals. Female mice (20-25 g) of Charles River strain (CD-i) were mated with the males of the same strain. The morning of finding the vaginal plug was designated as day 1 of pregnancy. Reverse Transcriptase Polymerase Chain Reaction (RTPCR). Amplification and detection of c-myc mRNA in preimplantation mouse embryos were achieved by RT-PCR. Embryos (about 80 per group) were collected from day 1-4 pregnant mice. Due to asynchronous embryonic developAbbreviations: oligo, oligodeoxyribonucleotide; EGF-R, epidermal growth factor receptor; RT-PCR, reverse transcriptase polymerase chain reaction. §To whom reprint requests should be addressed at: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS.
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Proc. Natl. Acad Sci. USA 89 (1992)
ment, a mixture of four-cell and eight-cell embryos was obtained on day 3 and late morulae and blastocysts on day 4. Embryos were washed several times to avoid any contamination with maternal cells and quick frozen in a small volume (5 LI) of culture medium in the bottom of a 400-.lI microcentrifuge tube. After addition of Escherichia coli rRNA (20 ,ug) to the tube, total RNA was extracted from these embryos using sodium dodecyl sulfate/phenol/chloroform buffers (39). c-myc mRNA was reverse transcribed and amplified by PCR as described (39, 40). Oligos (21 bases long) were synthesized based on the sequence of the mouse c-myc gene (41) as follows: P1 5'-CCTCTTCTCCACAGACACCAC-3'; P2 and P2S 5'-ATGCCCCTCAACGTGAACTTC-3l; P3 5'-CCCTATTTCATCTGCGACGAG-3'. The antisense oligo termed P1 was complementary to the c-myc gene beginning at base 53 relative to the start of exon 3 (Fig. la). P1 served as a primer for reverse transcription of c-myc mRNA and as the 3' primer during PCR. The sense oligos termed P2 and P2s began at the c-myc translation initiation codon in exon 2 and spanned the first seven codons (Fig. la). P2 served as the 5' primer during PCR. The sense oligo termed P3 began at codon 21 in exon 2 and was thus internal to the PCR primers (Fig. la). P3 was 5' end-labeled with 32p using T4 kinase, and the labeled primer was used to detect c-myc PCR a
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FIG. 1. RT-PCR, amplification, and detection of c-myc mRNA in preimplantation mouse embryos on days 1-4 of pregnancy. About 80 embryos per group were used for each day of pregnancy. (a) Structure of the mouse c-myc gene indicating the locations of the oligo primers (P1.3) used for RT-PCR and/or in embryo culture experiments. bp, Base pairs. (b) Southern blot detection of RT-PCR products specific for amplification of c-myc mRNA in preimplantation embryos obtained on the indicated days of pregnancy. c-myc transcripts were reverse transcribed into cDNA and then amplified using PCR (45 cycles). RT-PCR was predicted to amplify a 398-bp fragment of c-myc mRNA. Control PCR reactions containing RNA that had not been reverse transcribed were performed in parallel and yielded negative results. The control (lane C) represents day 4 embryo RNA. RT-PCR products were separated by 2% agarose gel electrophoresis, Southern blotted to nylon membranes, and hybridized to end-labeled P3. This probe is complementary to an internal region of the predicted c-myc RT-PCR product. Hybrids were detected by autoradiography.
products by Southern blot hybridization. c-myc transcripts were reverse transcribed into cDNA and then amplified using PCR (45 cycles) as described by Andrews et al. (39). The annealing temperature during PCR was 600C. Control PCR reactions containing RNA that had not been reverse transcribed were performed in parallel and yielded negative results. RTPCR products, equivalent to that obtained from about one embryo, based on the original sample size, were separated by 2% agarose gel electrophoresis, Southern blotted to nylon membranes, and hybridized at 500C to end-labeled P3 as described (42). Hybrids were detected by autoradiography. Sizes of RT-PCR products were estimated by comparison with a 123-bp DNA ladder. The predicted size of the RT-PCR product of c-myc mRNA was 398 bp. This experiment was repeated twice using independent isolates of preimplantation embryo RNA, and similar results were obtained. P2 and its complementary antisense oligo (sequence not shown) were unmodified, containing phosphodiester bonds and are referred to as sense c-myc and antisense c-myc, respectively. P2s and its complementary antisense oligo were chimeric or phosphorothioate/phosphodiester bonds with phosphothioester bonds located between the first two and the last two bases. These are referred to in the text as modified sense c-myc and modified antisense c-myc, respectively. These primers were used in embryo culture experiments. Immunohiochemistry. Embryos were recovered from the reproductive tract in Whitten's medium. They were freed of zona pellucida by a brief exposure to a 0.5% Pronase solution in phosphate-buffered saline (PBS) and washed several times in Whitten's medium (43). Embryos were placed onto poly(Llysine)-coated slides by cytocentrifugation, air-dried for 30 min, and fixed. For localization of c-myc, embryos were fixed in cold methanol for 10 min or in 1% paraformaldehyde in PBS for 30 min. After hydration in PBS for 30 min, and blocking in 10%o normal goat serum, embryos were incubated overnight at 40C in anti-c-myc antibody at a 1:500 dilution in PBS with 0.3% Triton X-100 (44). For localization of epidermal growth factor receptor (EGF-R), embryos were fixed in 10%6 neutral buffered formalin for 10 min and washed in PBS. They were then incubated in blocking solution (10%o normal goat serum) for 10 min followed by incubation in rabbit polyclonal antibody to mouse liver EGF-R (50 I&g/ml) (45) for 24 hr at 4°C. Immunostaining was performed using a Histostain-SP kit (Zymed Laboratories). The kit used a biotinylated secondary antibody, a horseradish peroxidasestreptavidin conjugate, and a substrate chromogen mixture. After immunohistochemistry, embryos were poststained lightly with fast green for c-myc and hematoxylin for EGF-R. Red deposits indicated the sites of immunostaining. Control experiments consisted of incubation of embryos with normal rabbit serum or in the absence of the primary antibody. Embryo Culture. To study effects of antisense c-myc oligos on preimplantation embryo development, two-cell embryos on day 2 (0800-0900 hr) of pregnancy were recovered from several mice and pooled in Whitten's medium containing 0.3% bovine serum albumin. Embryos were washed four times and cultured in batches of 8-15 in microdrops (25 ,ul) of culture medium under silicon oil in an atmosphere of 5% C02/95% air at 37°C for 72 hr (43) in the presence or absence of unmodified or modified antisense and sense c-myc oligos or with duplexes of the unmodified oligos. Oligos were added at the beginning of the culture. To generate sense-antisense duplexes, equal amounts of the sense (P2) and antisense c-myc oligos were mixed, heated to 800C for 5 min, and allowed to slowly cool to 370C for 5 hr prior to their addition to the embryo culture medium. Embryo development was monitored every 24 hr. At the end of the culture, the number of embryos that developed to blastocysts was recorded and the number of cells per blastocyst was determined (43). In control cultures, >80% of the embryos developed into blas-
Proc. Natl. Acad. Sci. USA 89 (1992)
Developmental Biology: Paria et al. tocysts. Each experiment was repeated three or four times with a total of at least 40-50 embryos per experimental point. To determine c-myc protein and EGF-R in blastocysts cultured in the presence of c-myc antisense or sense oligos, eight-cell (day 3) embryos were cultured (43) with either the c-myc sense oligo (P2) or antisense c-myc (7.5 ,uM) for 24 hr. At the end of the culture, those embryos that had formed a blastocoel were freed of zona pellucida, cytospun onto glass slides, and processed for immunolocalization of c-myc and EGF-R as described above.
RESULTS Expression of the c-myc gene in preimplantAtin mouse embryos was documented by detection ofc-myc IhRNA using the RT-PCR (Fig. 1) and by detection of c-'Yc protein using immunohistochemistry (Fig. 2). A search of the GenBank data base for sequence identity with the c-myc primers used for RT-PCR (P1 and P2) indicated that no amplification of other known gene transcripts should occur under these reaction conditions. Amplification of a 398-bp product was predicted from the sequence of c-myc cDNA. The specific detection of this RT-PCR product by Southern blot hybridization using the internal primer P3 as a probe further ensured the specificity of the assay. This primer (P3) has no significant sequence identity with other members of the c-myc family. RT-PCR failed to amplify c-myc transcripts in RNA obtained from one-cell embryos (Fig. lb; day 1 of pregnancy). In contrast, low levels of c-myc RT-PCR products were detected using two-cell embryo RNA (day 2), and high levels of RT-PCR amplified c-myc transcripts were detected using four-cell/eight-cell embryo RNA (day 3) and morula/blastocyst RNA preparations b
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(day 4). These results were reproduced using two independent preparations of RNA. No attempt was made to obtain quantitative RT-PCR results. Immunohistochemical detection of c-myc showed an absence of staining in one-cell and two-cell embryos, low-intensity nuclear staining in four-cell embryos, and intense nuclear staining at the eight-cell and morula/ blastocyst stages (Fig. 2). Overall, these results suggest that expression of the c-myc gene may be initiated, albeit at low levels or only transiently, during activation of the zygotic genome after the first cleavage (two-cell). In contrast, c-myc expression is heightened and apparently constitutive after the third cleavage (eight-cell) and remains high during differentiation of morulae into blastocysts. That c-myc plays an important role in preimplantation development was suggested by the finding of severely attenuated blastocyst formation from two-cell embryos cultured in the presence of a low concentration (7.5 AuM) of the antisense c-myc oligo (Fig. 3). This antisense c-myc oligo was complementary to the region of c-myc mRNA beginning with the translation initiation codon and spanning the next seven amino acids (reverse complement of P2 in Fig. la). When included at comparable concentrations (7.5 A&M) in the culture medium, the sense c-mnyc oligo (P2) and the duplex of these. antisense and sense oligos had little effect on blastocyst formation. However, embryos cultured *ith only 4-fold higher concentrations of any of the single or double-strand unmodified c-myc oligos arrested dev#lppment before the blastocyst stage. These embryos arrikted at the two-cell or four-cell stage, whereas embryos cult!Ired With low levels of antisense the eight-cell/morula stage and c-myc arrested developme did not proceed to the blastocyst stage. This suggested the possibility that a nonspecific toxic effect of DNA in the culture C
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FIG. 2. Immunohistochemical localization of c-myc protein in preimplantation mouse embryos on days 1-4 of pregnancy. Embryos were cytospun onto poly(L-lysine)-coated slides and fixed in 1% paraformaldehyde in PBS. After hydration in PBS and blocking in 109% normal goat serum, embryos were incubated in anti-c-myc antibody (44). Blastocysts were counterstained with fast green. Blastocysts incubated in nonimmune rabbit serum did not show specific staining (data not shown). Red (dark) deposits in the nucleus indicate the site of immunoreactive c-myc. Embryonic developmental stages shown are one-cell (a), two-cell (b), four-cell (c), eight-cell (d), decompacted eight-cell (e), and blastocyst (f). (x400.) Decompaction was achieved by incubating embryos in calcium-free medium. ICM, inner cell mass; Tr, trophectoderm.
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FIG. 3. Development of preimplantation embryos cultured with c-myc oligos. Two-cell embryos were cultured in batches of 8-15 in 25-til microdrops of culture medium under silicon oil at 370C for 72 hr in the presence or absence of the indicated concentrations of sense, antisense, or sense-antisense duplex c-myc oligos (see Fig. la). In other experiments, the culture medium contained the indicated concentrations of modified antisense or sense c-myc oligos. These modified oligos had phosphothioesters located between the first two bases and the last two bases of the oligo. Oligos were added at the beginning of the culture. In control cultures, >80% of the embryos developed into blastocysts. Each experiment was repeated three or four times with a total of 40-50 embryos per experimental point.
medium could influence the results. However, it was also noted that in vitro development offreshly recovered eight-cell embryos to the blastocyst stage was specifically inhibited (36% blastocyst formation) when cultured in medium containing 7.5 ,uM antisense c-myc, whereas the same concentration of sense c-myc (P2) had little effect (78% blastocyst formation). Examination of blastocysts that developed in the presence of antisense c-myc showed the complete absence of c-myc protein in the cell nuclei, whereas blastocysts that developed in medium containing sense c-myc stained positively for the protein (Fig. 4a). The effects of antisense c-myc appeared to be specific, since the abundance of immunoreactive EGF-Rs in similarly treated blastocysts remained unaltered (Fig. 4b). Those blastocysts that developed during culture with the antisense c-myc were not examined for the potential to continue development. These results are consistent with the suggestion that antisense c-myc acts to specifically attenuate synthesis of this protein.
To further validate the antisense c-myc effects, experiments were carried out using chimeric phosphorothioate/ phosphodiester oligos. The inclusion of phosphothioesters in oligos has been reported to increase their stability and uptake (26). As shown in Fig. 3, essentially all embryos (98%) cultured in the presence of a low concentration (7.5 AM) of the modified antisense c-myc oligo failed to develop to the blastocyst stage from the two-cell stage. In contrast, those embryos cultured in 4-fold higher concentrations (30 AM) of the modified sense c-myc oligo developed to the blastocyst stage with a frequency (80-90%o) indistinguishable from that of embryos cultured in the absence of oligo.
DISCUSSION These data provide evidence that activation of the zygotic c-myc gene and accumulation of this nuclear protein coincides with a critical function ofthis protein particularly at the
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FIG. 4. c-myc protein and EGF-R in blastocysts cultured in the presence of c-myc antisense or sense oligos. Eight-cell embryos were cultured with either the unmodified c-myc sense oligo (P2) or antisense c-myc (7.5 ,uM) for 24 hr, and at the end of the culture blastocysts that had developed were cytospun onto poly(L-lysine)-coated slides. (a) Embryos were fixed in 1% paraformaldehyde in PBS and incubated in anti-c-myc antibody (44). Photomicrographs of c-myc immunostaining in representative blastocysts that had developed in the presence of sense (Left) or antisense c-myc (Right) are shown at x300. (b) Embryos were fixed in 10%1 neutral buffered formalin in PBS and incubated in anti-EGF-R antibody (50 Ag/ml) (45). Photomicrographs of EGF-R immunostaining in representative blastocysts that had developed in the presence of sense (Left) or antisense c-myc (Right) are shown at x300.
D-9vevelopmental Biology: Paria et al. eight-cell/morula stage of development of preimplantation embryos. Thus, c-myc is apparently involved in the differentiation and/or late cleavages of embryos. Although the data suggest that this protein may not be critical for the first few cleavages of the fertilized egg, it is conceivable that uptake and/or stability of the oligo may differ between the early cleavage stage embryos as well as the eight-cell/morula and blastocyst. A lack of detection of either the c-myc protein or mRNA in these early embryos could be attributed to the transient expression of this gene during each of the first two cleavages. However, this seems unlikely since the majority of cells stained positively for c-myc at later stages of embryonic development (morulae and blastocysts). Further investigation will be required to resolve this matter. The mechanism by which c-myc expression in the preimplantation embryo is regulated is not known. It is possible that growth factors such as EGF/transforming growth factor a of reproductive tract (42) or embryonic origin (40) may play a role. EGF has been shown to up-regulate c-myc expression in cultured cells (46). During embryonic development ofthe mouse, EGF receptors are first detected at the eight-cell stage (43), and this growth factor has beneficial effects on growth and differentiation of mouse embryos into blastocysts in vitro (43). However, the possibility that other growth factors may be involved should not be ignored. These studies establish that antisense oligos can be employed as an effective approach to attenuating gene function during early mammalian embryogenesis. Although direct microinjection of antisense RNAs or DNAs as a means of blocking specific gene expression in mammalian and nonmammalian oocytes and early embryos has met with success and failure (47-52), a recent attempt to alter P-glucuronidase gene expression in the preimplantation mouse embryo by the addition of antisense oligos to the culture medium failed (53). Therefore, the approach reported here may not be gerierally applicable. The success of this approach may be largely dependent on the gene being examined, and we hypothesize that the extremely short half-life of c-myc mRNA and protein (20-22) may account in a large part for the success of these experiments. However, the studies herein also suggest that the concentration and the structure of the antisense oligo influence its effects on embryogenesis. mouse
Special thanks are due to Dr. Rosemary Watt (Smith Kline & French) for providing antibodies to human c-myc and to Dr. Eileen Adamson (La Jolla Cancer Research Foundation) for providing polyclonal antibodies to purified mouse liver EGF-R. This study was supported, in part, by grants from the National Institute of Child Health and Human Development (HD12304) to S.K.D. and from the National Institute of Environmental Health Sciences (ES04725) to
G.K.A.
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12. Kaczmarek, L., Hyland, J. K., Watt, R., Rosenberg, M. & Baserga, R. (1985) Science 228, 1313-1315. 13. Kingston, R. E., Baldwin, A. S. & Sharp, P. A. (1984) Nature (London) 312, 280-282. 14. Bishop, J. M. (1983) Annu. Rev. Biochem. 52, 301-354. 15. Varmus, H. E. (1984) Annu. Rev. Genet. 18, 553-612. 16. Pfeifer-Ohlsson, S., Rydnert, J., Goustin, A. S., Larsson, E., Betshltz, C. & Ohlsson, R. (1985) Proc. NatI. Acad. Sci. USA 82, 5050-5054. 17. Downs, K., Martin, G. & Bishop, J. M. (1989) Genes Dev. 3, 860(-869. 18. Hirning, U., Schmid, P., Schulz, W. A., Kozak, L. P. & Hameister, H. (1989) Cell Diff. Dev. 27, 243-248. 19. Zimmerman, K. A., Yancopoulos, G. D., Collum, R. G., Smith, R. K., Kohl, N. E., Denis, K. A., Nau, M. M., Witte, 0. N., Toran-Allerand, D., Gee, C. E., Minna, J. D. & Alt, F. W. (1986) Nature (London) 319, 780-783. 20. Blanchard, J. M., Piechaczyk, M., Dani, C., Chambard, J. C., Franchi, A., Pauyssegur, J. & Jeanteur, P. (1985) Nature (London) 317, 443-445. 21. Dony, C., Kessel, M. & Gruss, P. (1985) Nature (London) 317, 636-639. 22. Nishikura, K. & Murray, J. M. (1988) Oncogene 2, 493-498. 23. Weintraub, H. M., Izant, J. G. & Harland, R. M. (1985) Trends Genet. 1, 23-25. 24. Weintraub, H. M. (1990) Sci. Am. 262, 40-46. 25. Green, P. J., Pines, 0. & Inouye, M. (1986) Annu. Rev. Biochem. 55, 569-597. 26. Toulme, J. J. & Helene, C. (1988) Gene 72, 51-58. 27. van der Krol, A. R., Mol, J. N. M. & Stuitje, A. R. (1988) Biotechniques 6, 958-975. 28. Mutter, G. L. & Wolgemuth, D. J. (1987) Proc. Natl. Acad. Sci. USA 84, 5301-5305. 29. Propst, F., Rosenberg, M. P., Iyer, A., Kaul, K. & Vande Woude, G. F. (1987) Mol. Cell. Biol. 7, 1629-1637. 30. Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J. & Vande Woude, G. F. (1988) Nature (London) 335, 519-525. 31. Schmidt, M., Oskarsson, M. K., Dunn, J. K., Blair, D. G., Hughes, S. & Vande Woude, G. F. (1988) Mol. Cell. Biol. 8, 923-929. 32. Paules, R. S., Propst, F., Dunn, K. J., Blair, D. G., Kaul, K., Palmer, A. E. & Vande Woude, G. F. (1988) Oncogene 3, 59-68. 33. Herzog, N. K., Singh, B., Elder, T., Lipkin, I., Trauger, R. J., Millette, C. F., Goldman, D. S., Wolfes, H., Cooper, G. M. & Arlinghaus, R. B. (1990) Oncogene 3, 225-229. 34. Goldman, D. S., Kiessling, A. A. & Cooper, G. M. (1988) Oncogene 3, 159-162. 35. Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R. & Neckers, L. M. (1987) Nature (London) 328, 445-449. 36. Nishikura, K. & Murray, J. M. (1987) Mol. Cell. Biol. 1, 639-649. 37. Yokoyama, K. & Imamoto, F. (1987) Proc. Natl. Acad. Sci. USA 84, 7363-7367. 38. Holt, J. F., Render, R. L. & Nienhuis, A. W. (1988) Mol. Cell. Biol. 8, 963-973. 39. Andrews, G. K., Huet-Hudson, Y. M., Paria, B. C., McMaster, M. T., De, S. K. & Dey, S. K. (1991) Dev. Biol. 145, 13-27. 40. Rappolee, D. A., Brenner, C. A., Schultz, R., Mark, D. & Werb, Z. (1988) Science 241, 1823-1825. 41. Staton, L. W., Watt, R. & Marcu, K. B. (1983) Nature (London) 303, 401-406. 42. Huet-Hudson, Y. M., Chakraborty, C., De, S. K., Suzuki, S., Andrews, G. K. & Dey, S. K. (1990) Mol. Endocrinol. 4, 510-523. 43. Paria, B. C. & Dey, S. K. (1990) Proc. NatI. Acad. Sci. USA 87, 4756-4760. 44. Huet-Hudson, Y. M., Andrews, G. K. & Dey, S. K. (1989) Endocrinology 125, 1683-1690. 45. Weller, A., Meek, J. & Adamson, E. D. (1987) Development 100, 351-363. 46. Reuse, S., Roger, P. P., Vassart, G. & Dumont, J. E. (1986) Biochem. Biophys. Res. Commun. 141, 1066-1077. 47. Rebagliati, M. R. & Melton, D. A. (1987) Cell 48, 599-605. 48. Bass, B. L. & Weintraub, H. (1987) Cell 48, 607-613. 49. Strickland, S., Huart, J., Belin, D., Vassali, A., Rickles, R. J. & Vassali, J. D. (1988) Science 241, 680-684. 50. O'keefe, S. J., Wolfes, H., Kiessling, A. A. & Cooper, G. M. (1989) Proc. Natd. Acad. Sci. USA 86, 7038-7042. 51. Kimelman, D. & Kirschner, M. W. (1989) Cell 59, 687-698. 52. Bevilacqua, A., Loch-Caruso, R. & Erickson, R. P. (1989) Proc. NatI. Acad. Sci. USA 8, 5444-5448. 53. Ao, A., Erickson, R. P., Bevilacqua, A. & Karolyi, J. (1991) Antisense Res. Dev. 1, 1-10.
Antisense c-myc effects on preimplantation mouse embryo development BIBHASH C. PARIA*t, SUDHANSU K. DEY*t, AND GLEN K. ANDREWSt§ Departments of *Obstetrics-Gynecology, tPhysiology, and tBiochemistry and Molecular Biology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, KS 66103
Communicated by Clement L. Markert, July 6, 1992
ABSTRACT Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mehanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this
DNA-binding protein could be important during early embryogenesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containg an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eightcell/morula stage. At lower concentrations (7.5 FsM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 FM), all unmdified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 pM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with hih concentrations of the modified sense complement (30 FM). Culture of freshly recovered eight-cell embryos with antisene c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc fmction becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development.
The fertilized mammalian egg, after completion of several cleavages, differentiates into a blastocyst. The blastocyst is composed of the inner cell mass, which gives rise to the embryo proper and certain extraembryonic membranes, and the trophectoderm, which participates in the formation of extraembryonic structures (placenta and parietal yolk sac). Although activation of transcription of the embryonic genome occurs at the two-cell stage in the mouse (1, 2), our knowledge of genes that are crucial for preimplantation embryo development is meager. In this study, the role of c-myc in preimplantation mouse embryo development was examined. The c-myc protooncogene is the cellular homolog of the viral v-myc oncogene and is a member of a gene family that includes L-myc and N-myc (3-7). c-myc is a nuclear DNA-binding phosphoprotein that is highly conserved The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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throughout evolution. It is now thought that biological functions and sequence-specific DNA-binding activity of c-myc are executed by dimerization with another protein called Max (8-10). The expression of c-myc is positively correlated with DNA synthesis and cell proliferation (8-10) and is rapidly induced in various cell types after stimulation with mitogen (11). A direct role of c-myc in the cell cycle is suggested by the observation that quiescent cells enter the cell cycle and divide after microinjection of c-myc protein or transfection with the c-myc gene (12, 13). Because of the association of c-myc with cell cycle in cultured cells (14, 15) and the suggested role of this protein in cell division and differentiation during postimplantation mammalian development (1619), this protein could be important for cleavage and/or differentiation during early mammalian embryogenesis. Definitive proof for a role of c-myc in preimplantation embryo development requires examination of the effects of selective ablation of expression of this gene. Because of the extremely short half-life of c-myc mRNA and protein (20-22), the use of an antisense oligodeoxyribonucleotide (oligo) to c-myc mRNA to block its translation appeared an attractive approach toward elucidating the role ofthis protooncogene in preimplantation embryo development. Since the discovery of antisense RNAs as natural suppressors of prokaryotic genes (23-27), the successful use of antisense RNA/DNA to manipulate expression of endogenous and exogenous eukaryotic genes has been reported (28-34). Furthermore, several laboratories have reported inhibition of cell proliferation resulting from antisense blockage of expression of c-myc during culture in medium containing antisense oligo (35-38). MATERIALS AND METHODS Materials. HPLC-purified modified and unmodified sense and antisense oligos were purchased from Genosys, Houston. Rabbit polyclonal antibodies to human c-myc were kindly provided by Rosemary Watt (Smith Kline & French). Crystalline bovine serum albumin was purchased from Sigma (A-4378). DNA ladder was obtained from Bethesda Research Laboratories. All other reagents were purchased from Sigma, Fisher, or Mallinckrodt. Animals. Female mice (20-25 g) of Charles River strain (CD-i) were mated with the males of the same strain. The morning of finding the vaginal plug was designated as day 1 of pregnancy. Reverse Transcriptase Polymerase Chain Reaction (RTPCR). Amplification and detection of c-myc mRNA in preimplantation mouse embryos were achieved by RT-PCR. Embryos (about 80 per group) were collected from day 1-4 pregnant mice. Due to asynchronous embryonic developAbbreviations: oligo, oligodeoxyribonucleotide; EGF-R, epidermal growth factor receptor; RT-PCR, reverse transcriptase polymerase chain reaction. §To whom reprint requests should be addressed at: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS.
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Proc. Natl. Acad Sci. USA 89 (1992)
ment, a mixture of four-cell and eight-cell embryos was obtained on day 3 and late morulae and blastocysts on day 4. Embryos were washed several times to avoid any contamination with maternal cells and quick frozen in a small volume (5 LI) of culture medium in the bottom of a 400-.lI microcentrifuge tube. After addition of Escherichia coli rRNA (20 ,ug) to the tube, total RNA was extracted from these embryos using sodium dodecyl sulfate/phenol/chloroform buffers (39). c-myc mRNA was reverse transcribed and amplified by PCR as described (39, 40). Oligos (21 bases long) were synthesized based on the sequence of the mouse c-myc gene (41) as follows: P1 5'-CCTCTTCTCCACAGACACCAC-3'; P2 and P2S 5'-ATGCCCCTCAACGTGAACTTC-3l; P3 5'-CCCTATTTCATCTGCGACGAG-3'. The antisense oligo termed P1 was complementary to the c-myc gene beginning at base 53 relative to the start of exon 3 (Fig. la). P1 served as a primer for reverse transcription of c-myc mRNA and as the 3' primer during PCR. The sense oligos termed P2 and P2s began at the c-myc translation initiation codon in exon 2 and spanned the first seven codons (Fig. la). P2 served as the 5' primer during PCR. The sense oligo termed P3 began at codon 21 in exon 2 and was thus internal to the PCR primers (Fig. la). P3 was 5' end-labeled with 32p using T4 kinase, and the labeled primer was used to detect c-myc PCR a
0
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Exon 3
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P1
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369\
4w
246
123
C
l
1
2 3 4 Day of Pregnancy
FIG. 1. RT-PCR, amplification, and detection of c-myc mRNA in preimplantation mouse embryos on days 1-4 of pregnancy. About 80 embryos per group were used for each day of pregnancy. (a) Structure of the mouse c-myc gene indicating the locations of the oligo primers (P1.3) used for RT-PCR and/or in embryo culture experiments. bp, Base pairs. (b) Southern blot detection of RT-PCR products specific for amplification of c-myc mRNA in preimplantation embryos obtained on the indicated days of pregnancy. c-myc transcripts were reverse transcribed into cDNA and then amplified using PCR (45 cycles). RT-PCR was predicted to amplify a 398-bp fragment of c-myc mRNA. Control PCR reactions containing RNA that had not been reverse transcribed were performed in parallel and yielded negative results. The control (lane C) represents day 4 embryo RNA. RT-PCR products were separated by 2% agarose gel electrophoresis, Southern blotted to nylon membranes, and hybridized to end-labeled P3. This probe is complementary to an internal region of the predicted c-myc RT-PCR product. Hybrids were detected by autoradiography.
products by Southern blot hybridization. c-myc transcripts were reverse transcribed into cDNA and then amplified using PCR (45 cycles) as described by Andrews et al. (39). The annealing temperature during PCR was 600C. Control PCR reactions containing RNA that had not been reverse transcribed were performed in parallel and yielded negative results. RTPCR products, equivalent to that obtained from about one embryo, based on the original sample size, were separated by 2% agarose gel electrophoresis, Southern blotted to nylon membranes, and hybridized at 500C to end-labeled P3 as described (42). Hybrids were detected by autoradiography. Sizes of RT-PCR products were estimated by comparison with a 123-bp DNA ladder. The predicted size of the RT-PCR product of c-myc mRNA was 398 bp. This experiment was repeated twice using independent isolates of preimplantation embryo RNA, and similar results were obtained. P2 and its complementary antisense oligo (sequence not shown) were unmodified, containing phosphodiester bonds and are referred to as sense c-myc and antisense c-myc, respectively. P2s and its complementary antisense oligo were chimeric or phosphorothioate/phosphodiester bonds with phosphothioester bonds located between the first two and the last two bases. These are referred to in the text as modified sense c-myc and modified antisense c-myc, respectively. These primers were used in embryo culture experiments. Immunohiochemistry. Embryos were recovered from the reproductive tract in Whitten's medium. They were freed of zona pellucida by a brief exposure to a 0.5% Pronase solution in phosphate-buffered saline (PBS) and washed several times in Whitten's medium (43). Embryos were placed onto poly(Llysine)-coated slides by cytocentrifugation, air-dried for 30 min, and fixed. For localization of c-myc, embryos were fixed in cold methanol for 10 min or in 1% paraformaldehyde in PBS for 30 min. After hydration in PBS for 30 min, and blocking in 10%o normal goat serum, embryos were incubated overnight at 40C in anti-c-myc antibody at a 1:500 dilution in PBS with 0.3% Triton X-100 (44). For localization of epidermal growth factor receptor (EGF-R), embryos were fixed in 10%6 neutral buffered formalin for 10 min and washed in PBS. They were then incubated in blocking solution (10%o normal goat serum) for 10 min followed by incubation in rabbit polyclonal antibody to mouse liver EGF-R (50 I&g/ml) (45) for 24 hr at 4°C. Immunostaining was performed using a Histostain-SP kit (Zymed Laboratories). The kit used a biotinylated secondary antibody, a horseradish peroxidasestreptavidin conjugate, and a substrate chromogen mixture. After immunohistochemistry, embryos were poststained lightly with fast green for c-myc and hematoxylin for EGF-R. Red deposits indicated the sites of immunostaining. Control experiments consisted of incubation of embryos with normal rabbit serum or in the absence of the primary antibody. Embryo Culture. To study effects of antisense c-myc oligos on preimplantation embryo development, two-cell embryos on day 2 (0800-0900 hr) of pregnancy were recovered from several mice and pooled in Whitten's medium containing 0.3% bovine serum albumin. Embryos were washed four times and cultured in batches of 8-15 in microdrops (25 ,ul) of culture medium under silicon oil in an atmosphere of 5% C02/95% air at 37°C for 72 hr (43) in the presence or absence of unmodified or modified antisense and sense c-myc oligos or with duplexes of the unmodified oligos. Oligos were added at the beginning of the culture. To generate sense-antisense duplexes, equal amounts of the sense (P2) and antisense c-myc oligos were mixed, heated to 800C for 5 min, and allowed to slowly cool to 370C for 5 hr prior to their addition to the embryo culture medium. Embryo development was monitored every 24 hr. At the end of the culture, the number of embryos that developed to blastocysts was recorded and the number of cells per blastocyst was determined (43). In control cultures, >80% of the embryos developed into blas-
Proc. Natl. Acad. Sci. USA 89 (1992)
Developmental Biology: Paria et al. tocysts. Each experiment was repeated three or four times with a total of at least 40-50 embryos per experimental point. To determine c-myc protein and EGF-R in blastocysts cultured in the presence of c-myc antisense or sense oligos, eight-cell (day 3) embryos were cultured (43) with either the c-myc sense oligo (P2) or antisense c-myc (7.5 ,uM) for 24 hr. At the end of the culture, those embryos that had formed a blastocoel were freed of zona pellucida, cytospun onto glass slides, and processed for immunolocalization of c-myc and EGF-R as described above.
RESULTS Expression of the c-myc gene in preimplantAtin mouse embryos was documented by detection ofc-myc IhRNA using the RT-PCR (Fig. 1) and by detection of c-'Yc protein using immunohistochemistry (Fig. 2). A search of the GenBank data base for sequence identity with the c-myc primers used for RT-PCR (P1 and P2) indicated that no amplification of other known gene transcripts should occur under these reaction conditions. Amplification of a 398-bp product was predicted from the sequence of c-myc cDNA. The specific detection of this RT-PCR product by Southern blot hybridization using the internal primer P3 as a probe further ensured the specificity of the assay. This primer (P3) has no significant sequence identity with other members of the c-myc family. RT-PCR failed to amplify c-myc transcripts in RNA obtained from one-cell embryos (Fig. lb; day 1 of pregnancy). In contrast, low levels of c-myc RT-PCR products were detected using two-cell embryo RNA (day 2), and high levels of RT-PCR amplified c-myc transcripts were detected using four-cell/eight-cell embryo RNA (day 3) and morula/blastocyst RNA preparations b
10053
(day 4). These results were reproduced using two independent preparations of RNA. No attempt was made to obtain quantitative RT-PCR results. Immunohistochemical detection of c-myc showed an absence of staining in one-cell and two-cell embryos, low-intensity nuclear staining in four-cell embryos, and intense nuclear staining at the eight-cell and morula/ blastocyst stages (Fig. 2). Overall, these results suggest that expression of the c-myc gene may be initiated, albeit at low levels or only transiently, during activation of the zygotic genome after the first cleavage (two-cell). In contrast, c-myc expression is heightened and apparently constitutive after the third cleavage (eight-cell) and remains high during differentiation of morulae into blastocysts. That c-myc plays an important role in preimplantation development was suggested by the finding of severely attenuated blastocyst formation from two-cell embryos cultured in the presence of a low concentration (7.5 AuM) of the antisense c-myc oligo (Fig. 3). This antisense c-myc oligo was complementary to the region of c-myc mRNA beginning with the translation initiation codon and spanning the next seven amino acids (reverse complement of P2 in Fig. la). When included at comparable concentrations (7.5 A&M) in the culture medium, the sense c-mnyc oligo (P2) and the duplex of these. antisense and sense oligos had little effect on blastocyst formation. However, embryos cultured *ith only 4-fold higher concentrations of any of the single or double-strand unmodified c-myc oligos arrested dev#lppment before the blastocyst stage. These embryos arrikted at the two-cell or four-cell stage, whereas embryos cult!Ired With low levels of antisense the eight-cell/morula stage and c-myc arrested developme did not proceed to the blastocyst stage. This suggested the possibility that a nonspecific toxic effect of DNA in the culture C
zx'.!
./
di
e
FIG. 2. Immunohistochemical localization of c-myc protein in preimplantation mouse embryos on days 1-4 of pregnancy. Embryos were cytospun onto poly(L-lysine)-coated slides and fixed in 1% paraformaldehyde in PBS. After hydration in PBS and blocking in 109% normal goat serum, embryos were incubated in anti-c-myc antibody (44). Blastocysts were counterstained with fast green. Blastocysts incubated in nonimmune rabbit serum did not show specific staining (data not shown). Red (dark) deposits in the nucleus indicate the site of immunoreactive c-myc. Embryonic developmental stages shown are one-cell (a), two-cell (b), four-cell (c), eight-cell (d), decompacted eight-cell (e), and blastocyst (f). (x400.) Decompaction was achieved by incubating embryos in calcium-free medium. ICM, inner cell mass; Tr, trophectoderm.
10054
Developmental Biology: Paria et al.
Proc. Nad. Acad. Sci. USA 89 (1992)
100
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0 A
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0 0
40
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0
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30.00
FIG. 3. Development of preimplantation embryos cultured with c-myc oligos. Two-cell embryos were cultured in batches of 8-15 in 25-til microdrops of culture medium under silicon oil at 370C for 72 hr in the presence or absence of the indicated concentrations of sense, antisense, or sense-antisense duplex c-myc oligos (see Fig. la). In other experiments, the culture medium contained the indicated concentrations of modified antisense or sense c-myc oligos. These modified oligos had phosphothioesters located between the first two bases and the last two bases of the oligo. Oligos were added at the beginning of the culture. In control cultures, >80% of the embryos developed into blastocysts. Each experiment was repeated three or four times with a total of 40-50 embryos per experimental point.
medium could influence the results. However, it was also noted that in vitro development offreshly recovered eight-cell embryos to the blastocyst stage was specifically inhibited (36% blastocyst formation) when cultured in medium containing 7.5 ,uM antisense c-myc, whereas the same concentration of sense c-myc (P2) had little effect (78% blastocyst formation). Examination of blastocysts that developed in the presence of antisense c-myc showed the complete absence of c-myc protein in the cell nuclei, whereas blastocysts that developed in medium containing sense c-myc stained positively for the protein (Fig. 4a). The effects of antisense c-myc appeared to be specific, since the abundance of immunoreactive EGF-Rs in similarly treated blastocysts remained unaltered (Fig. 4b). Those blastocysts that developed during culture with the antisense c-myc were not examined for the potential to continue development. These results are consistent with the suggestion that antisense c-myc acts to specifically attenuate synthesis of this protein.
To further validate the antisense c-myc effects, experiments were carried out using chimeric phosphorothioate/ phosphodiester oligos. The inclusion of phosphothioesters in oligos has been reported to increase their stability and uptake (26). As shown in Fig. 3, essentially all embryos (98%) cultured in the presence of a low concentration (7.5 AM) of the modified antisense c-myc oligo failed to develop to the blastocyst stage from the two-cell stage. In contrast, those embryos cultured in 4-fold higher concentrations (30 AM) of the modified sense c-myc oligo developed to the blastocyst stage with a frequency (80-90%o) indistinguishable from that of embryos cultured in the absence of oligo.
DISCUSSION These data provide evidence that activation of the zygotic c-myc gene and accumulation of this nuclear protein coincides with a critical function ofthis protein particularly at the
b a
......,g E. ...
FIG. 4. c-myc protein and EGF-R in blastocysts cultured in the presence of c-myc antisense or sense oligos. Eight-cell embryos were cultured with either the unmodified c-myc sense oligo (P2) or antisense c-myc (7.5 ,uM) for 24 hr, and at the end of the culture blastocysts that had developed were cytospun onto poly(L-lysine)-coated slides. (a) Embryos were fixed in 1% paraformaldehyde in PBS and incubated in anti-c-myc antibody (44). Photomicrographs of c-myc immunostaining in representative blastocysts that had developed in the presence of sense (Left) or antisense c-myc (Right) are shown at x300. (b) Embryos were fixed in 10%1 neutral buffered formalin in PBS and incubated in anti-EGF-R antibody (50 Ag/ml) (45). Photomicrographs of EGF-R immunostaining in representative blastocysts that had developed in the presence of sense (Left) or antisense c-myc (Right) are shown at x300.
D-9vevelopmental Biology: Paria et al. eight-cell/morula stage of development of preimplantation embryos. Thus, c-myc is apparently involved in the differentiation and/or late cleavages of embryos. Although the data suggest that this protein may not be critical for the first few cleavages of the fertilized egg, it is conceivable that uptake and/or stability of the oligo may differ between the early cleavage stage embryos as well as the eight-cell/morula and blastocyst. A lack of detection of either the c-myc protein or mRNA in these early embryos could be attributed to the transient expression of this gene during each of the first two cleavages. However, this seems unlikely since the majority of cells stained positively for c-myc at later stages of embryonic development (morulae and blastocysts). Further investigation will be required to resolve this matter. The mechanism by which c-myc expression in the preimplantation embryo is regulated is not known. It is possible that growth factors such as EGF/transforming growth factor a of reproductive tract (42) or embryonic origin (40) may play a role. EGF has been shown to up-regulate c-myc expression in cultured cells (46). During embryonic development ofthe mouse, EGF receptors are first detected at the eight-cell stage (43), and this growth factor has beneficial effects on growth and differentiation of mouse embryos into blastocysts in vitro (43). However, the possibility that other growth factors may be involved should not be ignored. These studies establish that antisense oligos can be employed as an effective approach to attenuating gene function during early mammalian embryogenesis. Although direct microinjection of antisense RNAs or DNAs as a means of blocking specific gene expression in mammalian and nonmammalian oocytes and early embryos has met with success and failure (47-52), a recent attempt to alter P-glucuronidase gene expression in the preimplantation mouse embryo by the addition of antisense oligos to the culture medium failed (53). Therefore, the approach reported here may not be gerierally applicable. The success of this approach may be largely dependent on the gene being examined, and we hypothesize that the extremely short half-life of c-myc mRNA and protein (20-22) may account in a large part for the success of these experiments. However, the studies herein also suggest that the concentration and the structure of the antisense oligo influence its effects on embryogenesis. mouse
Special thanks are due to Dr. Rosemary Watt (Smith Kline & French) for providing antibodies to human c-myc and to Dr. Eileen Adamson (La Jolla Cancer Research Foundation) for providing polyclonal antibodies to purified mouse liver EGF-R. This study was supported, in part, by grants from the National Institute of Child Health and Human Development (HD12304) to S.K.D. and from the National Institute of Environmental Health Sciences (ES04725) to
G.K.A.
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