The Bacteriophage T7 Binary System Activates Transient Transgene Expression in Zebrafish (Danio rerio) Embryos

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

237, 492–495 (1997)

RC977170

The Bacteriophage T7 Binary System Activates Transient Transgene Expression in Zebrafish (Danio rerio) Embryos Tiziano Verri,* Francesco Argenton,† Rosella Tomanin,‡ Maurizio Scarpa,‡ Carlo Storelli,* Rodolfo Costa,* Lorenzo Colombo,† and Marino Bortolussi†,1 †Department of Biology, University of Padua; *Department of Biology, University of Lecce; and ‡CRIBI Biotechnology Centre and Department of Pediatrics, University of Padua, Italy

Received July 18, 1997

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model. q 1997 Academic Press

The bacteriophage T7 binary expression system is widely used for the in vitro expression of RNAs and proteins. It consists of T7 RNA polymerase (T7pol), the product of gene 1, and the promoter and terminator of gene 10 (1). T7pol is a monomeric enzyme of about 100 kDa that is able to elongate RNA molecules about five times faster than Escherichia coli RNA polymerase (1). T7pol recognizes with high specificity its own promoter, consisting of a 23 bp-long sequence which does not occur in other prokaryotic and eukaryotic promoters (2). Such a high catalytic activity and strong promoter specificity make the enzyme very useful for the in vitro expression of target proteins in both prokaryotic and eukaryotic cells. The two elements of the system have 1 Corresponding author: Dipartimento di Biologia, Universita` di Padova, Via U. Bassi 58/B, I-35121 Padova, Italy. Fax: /39 49 8276344. E-mail: [email protected].

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first been cloned into plasmids (3) and subsequently introduced into vaccinia virus (4), baculovirus (5) and, recently, adenovirus (6) vectors. However, to our knowledge, the possibility to apply the T7 binary system in an in vivo setting has not yet been investigated. The zebrafish (Danio rerio) transient expression system represents a convenient and reproducible model to study the expression of foreign genes in vivo (7). In this system, heterologous genes can be expressed in developing embryos after microinjection of plasmid DNA into fertilized eggs. The zebrafish model was also shown to utilize effectively using eukaryotic binary systems in which a transcription factor produced by an effector plasmid transactivates a promoter sequence driving a reporter gene in a second plasmid (8,9). In the present study, we have used the zebrafish assay to investigate whether the T7 binary system could work in an in vivo setting. To this purpose, zebrafish fertilized eggs were microinjected with two plasmids carrying separately the two elements of the T7 binary system. We show that the T7 system operates efficiently in 24-h mosaic transgenic zebrafish embryos, suggesting its potentiality for the in vivo expression of selected RNAs or proteins in vertebrate models. MATERIALS AND METHODS Plasmids. pE1T7R (6) carries the bacteriophage T7 RNA polymerase gene, provided with the 36 bp-oligonucleotide sequence encoding the SV40 nuclear location signal (NLS) (10), driven by the cytomegalovirus (CMV) promoter, and followed by the SV40 polyadenylation signal. pT7EMNLSlacZ and pT7EMNLSlacZpolyA (6) carry the E. coli lacZ gene, encoding b-galactosidase (bgal), driven by the T7 promoter and followed by the T7 terminator. In the latter plasmid, the SV40 polyadenylation signal was added 5* of the T7 terminator. In both plasmids, the short untranslated region of the encephalomyocarditis virus (EMCV-UTR) (11) is situated downstream of the T7 promoter. The cloning steps leading to the construction of the 3 vectors described above are reported in details elsewhere (6). The positive (pCMV-bgal) (12) and negative (pCMV-Luc) (8) control plasmids, containing the CMV promoter driving the lacZ and the firefly luciferase (Luc) genes, respectively, have been already described.

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FIG. 1. Schematic maps of the plasmids used in this study. In the pE1T7R expression plasmid: CMV promoter, cytomegalovirus promoter; NLS, SV40 nuclear location signal. In the pT7EMNLSlacZ and pT7EMNLSlacZpolyA reporter plasmids: EMCV, untranslated region of the encephalomyocarditis virus (EMCV-UTR); lacZ, gene encoding b-galactosidase.

Microinjection of zebrafish embryos. All solutions for microinjection were prepared by diluting plasmid DNA to a final concentration of 50 ng/ml in PBS, pH 7.3. Phenol red (0.25%) was also included to monitor the course of injection. Zebrafish were raised and bred according to standard procedures (13). Immediately after spawning, the eggs were collected by siphoning the bottom of the aquaria and placed into round grooves which were produced in agarose plates using 1-mm glass capillaries (8). Fertilized eggs (one- to four-cell stage) were injected with approximately 0.3 nl of DNA solution containing either pE1T7R or pCMV-Luc and one of the bgal reporter plasmids (pT7EMNLSlacZ or pT7EMNLSlacZpolyA) in a 1:1 proportion. Positive control embryos were injected with pCMV-bgal alone. After 24 h incubation at 287C, the embryos were fixed 30 min at 47C in a solution containing: 4% paraformaldehyde, 0.2% glutaraldehyde, 4% sucrose, 0.15 mM CaCl2 , 0.16 mM Na2HPO4 , and 0.04 mM NaH2PO4 , pH 7.3. Localization of bgal activity was performed using X-Gal staining, as described elsewhere (7), with minor modifications. After 24 h staining, embryos were fixed again for 2 h at room temperature, dechorionated, mounted in glycerol/PBS 1:1 and stained. Cell counts were performed at 4001 magnification.

RESULTS The plasmids used in the present investigation are depicted schematically in Fig. 1. The T7 binary system is composed by the pE1T7R expression vector in combination with either the pT7EMNLSlacZ or the pT7EMNLSlacZpolyA reporter plasmids. In pE1T7R, the CMV promoter drives the expression of the T7pol gene provided with the SV40 NLS that targets T7pol to the nucleus and allows the transcription of T7 promoterdriven sequences in an eukaryotic environment (10). In pT7EMNLSlacZ and pT7EMNLSlacZpolyA, the T7 promoter, which is fused to the EMCV-UTR to allow cap-independent translation of mRNA (11), drives the

expression of the lacZ gene. In addition, pT7EMNLSlacZpolyA carries the SV40 polyadenylation signal which has been reported to improve significantly the efficiency of the T7pol binary system in vitro (14). As revealed histochemically, microinjection into zebrafish fertilized eggs of the pCMV-bgal plasmid, representing the positive control of both CMV promoter activity and reporter function, resulted in a mosaic pattern of expression of bgal in about 80% of 24-h embryos (Fig. 2A, Table 1). This confirms earlier results (7,9) and is due to both the random distribution of the injected plasmid within the embryo and to the ability of the CMV promoter to activate transcription ubiquitously. Coinjection of pE1T7R in combination with either pT7MNLSlacZ or pT7MNLSlacZpolyA also resulted in the mosaic expression of the marker gene in a similar percentage of 24-h embryos (Fig. 2B, Table 1). Moreover, the average number of stained cells (from 40 to 50) did not differ significantly in both embryos injected with pCMV-bgal and those receiving either combinations of plasmids comprising the T7 system (Table 1). These results demonstrate the effectiveness of the T7 binary system to transactivate a target gene in the zebrafish model, and also show that the efficiency of the reporter plasmid was not improved by the addition of the SV40 polyadenylation signal. Conversely, most embryos coinjected with pCMV-Luc instead of pE1T7R lacked bgal activity (Fig. 2C), and those in which the marker gene was detected contained only one or two stained cells (Table 1), indicating that significant activation of the T7 promoter in zebrafish embryos required T7pol. Under our experimental conditions, survival of embryos was unaffected and obvious malformations did not appear, indicating that injection of the plasmids and expression of the T7pol did not interfere with normal embryonic development. DISCUSSION Our investigation shows that microinjection of the two components of the T7 expression system into zebrafish fertilized eggs results in an efficient expression of the reporter gene in 24-h mosaic transgenic embryos. This demonstrates that the T7 system may be used, as other binary expression systems such as the yeast Gal4/UAS (15,16) or E. coli tetracycline-responsive system (17,18), to achieve the in vivo production of target RNAs and proteins. However, in the T7 system, general transcription factors are only needed for the production of the enzyme driven by the CMV promoter, since T7pol alone is sufficient to transactivate the T7 promoter. Therefore, the T7 system requires conceivably much less transcription factors than the above-mentioned systems and is thus expected to interfere less significantly with overall transcription in the host cells. In this respect, it should be noted that, in our experiments, the T7-driven expression of the reporter gene

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FIG. 2. b-Galactosidase activity in 24-h zebrafish embryos injected with (A) pCMV-bgal, (B) pE1T7R plus pT7EMNLSlacZpolyA, and (C) pCMV-Luc plus pT7EMNLSlacZpolyA. Results obtained with pT7EMNLSlacZ were similar to those obtained with pT7EMNLSlacZpolyA.

did not affect the normal development of embryos. The high catalytic activity of T7pol represents a further advantage because a minimal quantity of the enzyme might be sufficient to obtain a proper expression of the target gene driven by the T7 promoter, as shown in vitro (6). On the other hand, a T7 autogene construct, in which the T7 promoter combined with the CMV promoter controls T7 pol transcription, could be used to increase T7 pol expression without further interference on the host cell transcription. In the absence of T7pol, the reporter plasmids were

practically inactive, as only an occasional stained cell was encountered in some of the embryos coinjected with pCMV-Luc instead of the pE1T7R. The stringent necessity of T7pol to obtain significant expression from the T7 promoter had been also demonstrated in vitro using adenovirus vectors expressing the T7 system (6). The very limited in vivo activity of the T7 promoter in the absence of T7pol is relevant to its possible application for the targeted expression of genes encoding toxic proteins in transgenic animals. This may be useful to test the effects of selective destruction of specific cell

TABLE 1

Quantitative Analysis of Embryos and Cells Expressing b-Galactosidase Injected plasmids

Injected embryos

pCMV-bgal pE1T7R / pT7EMNLSlacZpolyA pCMV-Luc / pT7EMNLSlacZpolyA pE1T7R / pT7EMNLSlacZ pCMV-Luc / pT7EMNLSlacZ

90 129 47 92 36

a

Positive embryosa 71 114 6 73 4

(78.9%) (88.4%) (12.8%) (79.3%) (11.1%)

Embryos containing at least one stained cell. 494

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Stained cells/ positive embryo 48.8 53.7 1.3 41.6 1.5

{ { { { {

5.2 9.1 0.2 12.5 0.4

(nÅ19) (nÅ28) (nÅ6) (nÅ12) (nÅ4)

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types or tissues in an in vivo context. To this purpose, transgenics bearing a gene encoding a toxic protein driven by the T7 promoter could be mated with other transgenics carrying the T7pol gene driven by a cell- or tissue-specific promoter. Moreover, the high catalytic activity of T7pol could also be exploited to generate transgenic animals which express antisense RNA aimed at suppressing target mRNA translation. ACKNOWLEDGMENTS This work was supported by grants from the Italian Ministry of Agriculture, Food, and Forestry (Fourth Plan on Fisheries and Aquaculture in Marine and Brackish Waters) and the National Research Council of Italy (Grant 95.01943.CT14).

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5. van Poelwijk, F., Broer, R., Belsham, G. J., Oudshoorn, P., Vlak, J. M., and Godbach, R. W. (1995) Bio/Technology 13, 261–264. 6. Tomanin, R., Bett, A. J., Picci, L., Scarpa, M., and Graham, F. L. (1997) Gene, in press. 7. Westerfield, M., Wegner, J., Jegalian, B. G., DeRobertis, E. M., and Puschel, A. W. (1992) Genes Dev. 6, 591–598. 8. Argenton, F., Arava, Y., Aronheim, A., and Walker, M. D. (1996) Mol. Cell. Biol. 16, 1714–1721. 9. Argenton, F., Walker, M. D., Colombo, L., and Bortolussi, M. (1997) FEBS Lett. 407, 191–196. 10. Dunn, J. J., Krippl, B., Bernstein, K. E., Westphal, H., and Studier, F. W. (1988) Gene 68, 259–266. 11. Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) Proc. Nat. Acad. Sci. USA 86, 6126–6130. 12. MacGregor, G. R., Mogg, A. E., Burke, C. T., and Caskey, C. T. (1987) Somat. Cell Mol. Genet. 13, 253–265. 13. Westerfield, M. (1995) The Zebrafish Book, a Guide for the Laboratory Use of Zebrafish, 3rd ed., Univ. Oregon Press, Eugene, OR. 14. Deng, H., Wang, C., Acsadi, G., and Wolff, J. A. (1991) Gene 109, 193–201. 15. Ornitz, D. M., Moreadith, R. W., and Leder, P. (1991) Proc. Nat. Acad. Sci. USA 88, 698–702. 16. Kaiser, K. (1993) Curr. Biol. 3, 560–562. 17. Furth, P. A., St. Onge, L., Bo¨ger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Hennighausen, L. (1994) Proc. Nat. Acad. Sci. USA 91, 9302–9306. 18. Yu, Z. H., Redfern, C. S., and Fishman, G. I. (1996) Circ. Res. 79, 691–697.

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