32P by nick translation. After hybridization, the filters were washed and exposed to X. AR Kodak film. RNA analysis. RNAs were prepared by the hot phenol ...
Volume 16 Number 5 1988
Nucleic Acids Research
Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line Catherine Cavard, Gis6le Grimber, Nathalie Dubois, Jean-Fran4ois Chasse, Myriam Bennoun, Michele Minet-Thuriaux, Pierre Kamoun and Pascale Briand
Laboratoire de Biochimie Gdn6tique, H6pital Necker-Enfants Malades, 149 Rue de Sevres, 75743 Paris Cddex, France Received October 28, 1987; Revised December 23, 1987; Accepted January 15, 1988
ABSTRACT The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 earlv promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated
INTRODUCTION mice have been shown to be useful for investigating Transgenic regulation of gene expression and dissociating complex biological processes. They can also serve as animal models for some human diseases and for the correction of genetic defects in mice (1) . Recently, transgenic animals have been obtained using recombinant retrovirus techniques (2), but the classical method is by microinjection of the fo:^eign gene into one of the pronuclei of a fertilized mouse embryo (3). We have used this method to attempt a correction of the mouse Spf-ash ornithine transcarbamylase (OTC) deficiency. OTC is a mitochondrial matrix enzyme that catalyzes the synthesis of citrulline from ornithine and carbamylphosphate in the mammalian urea cycle . The enzyme is encoded by an X-linked gene (4) and is expressed in the liver and small intestine (5). Human OTC deficiency is one of the most frequent hereditary hyperammonemias
© I R L Press Limited, Oxford, England.
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Nucleic Acids Research (6). This X-linked inherited disease leads to the death of 75% of affected males whatever the treatment. Thus although several types of mutation leading to various levels of residual activity have been described, the course is in most cases very dramatic (7). Two strains of mice with an OTC mutation have been described: sparse-fur (Spf) (8) and sparse fur with abnormal skin and hair (Spf-ash) (9). The Spf mouse has an OTC deficiency associated with decreased OTC activity, a change in optimal pH, altered substrate affinity and an increased amount of cross-reacting material reactive with an antibody to OTC (10). A C to A transversion 348 bp downstream from the translation initiation point has been recently shown to be the orloin of this deficiency and results in the replacement of a His residue with an Asn residue at amino acid position 117 (11). The Spf-ash OTC deficiency is characterized by a parallel reduction in the amounts of OTC activity, OTC protein and specific OTC mRNA to 5% of control values (12). In these two strains, there is a hyperammonemia, a pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. The reason why mouse OTC deficiencies lead to this sparse fur phenotype is unknown. These two allelic mutations constitute suitable models of hereditary hyperammonemia and more specifically of partial OTC deficiencies in man (12). We have used the Spf-ash strain to attempt a correction of the enzymatic defect by gene transfer. The molecular basis of the Spf-ash mutation has not been fully elucidated but we have demonstrated that mitochondria isolated from the liver of these mutants were able to correctly transport and mature a normal OTC pre-enzyme (12). Thus , the introduction in the Spf-ash embryos of a sequence able to direct the synthesis of a normal OTC preenzyme could allow the production of a mature and active OTC in the mitochondria.
MATERIALS AND METHODS Preparation of rat OTC cDNA for microinjection. A Xba I / Nsi I DNA fragment, 1.2 kilobases (kb) long, containing the rat OTC cDNA was excised from a recombinant plasmid pMN 152 (13) kindly supplied by G.SHORE. This fragment was introduced between the
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Nucleic Acids Research Apa I and Hind III restriction sites of the pBB3 plasmid (14) in replacement of the Eco gpt gene. The entire plasmid pBB3 OTC (4.95 kb long) was linearized by Eco RI and diluted in 10 mmol/l Tris-HCl pH 8.0, 0.1 mmol/l EDTA, to obtain approximately 100 copies per pl. Production of transRenic mice. Homozygous Spf-ash females were mated with C57BL/6J x DBA males. Fertilized eggs were isolated from oviducts , freed from follicle cells by hyaluronidase treatment and microinjected as dezzribed by Gordon and Ruddle (15) About 1 pl of the DNA solution was microinjected in one of the pronuclei. The eggs were then transferred to the oviducts of pseudopregnant mice. DNA analysis. DNA was prepared from tail biopsy samples from 2week-old mice. The presence of the rat cDNA was checked b.y Southern blot analysis using 15 pg of DNA previously digested by either Hind III, Eco RI or Pst I restriction enzymes. The digests were separated on 0.8% agarose gels by horizontal electrophoresis at 1.5 V/cm for 16 h. DNA was denatured in situ and transferred to a Zetabind membrane ac:cording to the supplier's recommendations. The DNA probe was either pBB3 OTC, pBB3 or the Apa I/Bam HI fragment of pBB3, labelled with dCTP- 32 P by nick translation After hybridization, the filters were washed and exposed to X AR Kodak film. RNA analysis. RNAs were prepared by the hot phenol procedure (16) . Briefly, tissues were pulverized in liquid nitrogen and submitted to 3 phenol extractions in 10 mmol/l sodium acetate pH 5.2, 0.5% SDS, at 55 C. After a final extraction by chloroform/ isoamylalcohol, RNAs were precipitated by addition of 2.5 volumes of ethanol and 0.1 volume of 3 mol/l sodium acetate pH 7.2. They were separated on 1.2% agarose formaldehyde gels by horizontal electrophoresis at 2 V/cm. They were transferred to a Zetabind membrane as above. The probe used was pBB3 OTC labelled by nick translation with dCTP- 32p. Orotic acid quantification. Orotic aciduria was determined as described (17). Ornithine transcarbamylase assay. Tissue samples were immediatelyhomogenized and OTC activity determined as described (18).
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truction. The pBB3 OTC plasmid contains the SV40 early promoter directly linked to the rat OTC cDNA which is followed by two fragments of SV40 DNA. The first is the small t-antigen intron and the second contains the viral early polyadenylation signal. pML2 is a pBR322 derivative that was deleted for sequences that Interfere with replication in eucaryotic cells.
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The en,tire rat OTC cDNA was excised from pMN152, thus eliminating the poly-dCdG clon-ing tails, and directly linked to the SV40 early promoter (Fig 1). Approxinatelv 100 copies of the resulting plasmid pBB3 OTC linearized by Eco RI, were microinjected into fertilized eggs obtained from homozygous Spf-ash females mated with C57BL males. With this mating, the eggs obtained by superovulation have easily detectable pronuclei providing good material for microinjection. On the other hand, males are hemizygous and have the abnormal phenotype previously descri-bed and females are heterozygous with a normal phenotype. Among the 120 eggs microinjected, 85 su-rvived and were reintroduced into foster mothers. Twenty-one newborns were obtained. In one litter composed of only two males, one had a normal phenotype suggesting that the OTC transgene was present and expressed (Fig 2).
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Nucleic Acids Research Integration and transmission to the progeny. Hind III digests of DNA isolated from the tails of various controls and from the supposed transgenic animal were analysed by Southern Blot. Hybridithe in zation with the Apa I/Bam HI fragment of pBB3 reveals transgenic animal, a 1.2 kb band which is not detected either in the spf-ash/spf-ash and spf-ash/+ females or in the spf-ash/Y and +/Y males (Fig 3 A). This demonstrates that at least a portion of the exogenous DNA sequence introduced is present. Its size is not compatible with a classical multicopy insertion in tandem array Transmission of the transgene to of an unrearranged sequence the progeny was also investigated. The number of transgenic animals in the Fl generation is compatible with a mendelian transmission (Fig 4 ), but the differences observed between the Hind III patterns of the founder and its progeny suggest a more complex transmission process. In fact, in the progeny, hybridization with the pBB3 plasmid which contains the Apa I/Bam HI probe used to characterize the founder reveals a single 7 kb band (Fig 3A). 1- a single Two hypotheses may account for this observation : in the germ transgene insertion followed by r'earrangement of the cells of the founder, as has been previously described by Palmi2- a mosaicisn, in the founder with a heterogeter et al (19) nous distribution of two integrated sequences; this is not frequent but has already been described by Wagner et al (20). Analysis of the remaining restriction sites suggests that deletions at the 5' and 3' ends of the sequence present in the progeny have occurred as often happens when a transgene is introduced by miIn the following generations, the DNA pattern croinjection (21) is stable (Fig 3) and its supposed structure is shown in Fig 5. .
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When adequate matings are performed between the transgenic animal and spf-ash females, the transmission of the transgene is always Figure 3. Southern blot analysis of the tail DNA trom the transgenic animal (tr), three of its progeny (Fll, F12, F21) and of controls Spf-ash/Spf-ash, Spf-ash/+, Spf-ash/Y, +/Y 15 pg of DNA were digested by Hind III (A), Eco RI (B), Pst I (C, D). Membranes were hybridized with the following probes: the 850 bp Apa IBam HI fragment of pBB3 for (A), pBB3 for (B) and (C) and pBB3 OTC for (D). Molecular sizes are expressed in kb and indicated by arrows.
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Nucleic Acids Research
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Filzure 6. Northern blot analysis of pOTC mRNA from various tissues of normal and transgenic Spf-ash/Spf-ash inice. RNA (10 yg) were fractionated on a 1.5% agarose/formaldehyde ,.el and trans-
ferred to a Zetabind membrane. The filter was prooed with 32p_ labelled OTC cDNA and autoradiographed for 2 days. A longer exposure time was performed to check the lack of pOTC mRNA in tissues except liver and small intestine. The positions of mouse 18 S and 28 S ribosomal markers are indicated by arrows.
follows the pyrimidine pathway. The saturation of this metabolic pathway induces orotic aciduria which in the Spf-ash animals reaches 20 umol/mg creatinine . In the transgenic animal orotic aciduria was reduced to a quite normal value (1.4 umol/mg creatinine versus 0.25 umol/mg in controls). Direct evidence of the transgene expression was obtained in the transgenic Spf-ash animals of the Fl. High levels of OTC activity (80 to 90% of control values) were found in the liver and small intestine, the normal sites of endooenous OTC activity. lqe also detected an OTC expression in the spleen and lung ( respectively 0.15% and 0.30X0 of the hepatic value) where activity has never been found in normal or mutant mice. 2107
Nucleic Acids Research Transgene expression was also studied at the RNA level. As expected, Northern blot analysis did not detect any difference between normal and transgenic mRNA lenoth at the resolution used (Fio 6). Parallel hybridizations of dot blots with pBB3 OTC and poly-U allowed quantification of OTC mRNA levels ( data not showvn): the livers of transgenic animals contained only 50% of OTC mRNA compared to controls while the enzymatic activity was 80%. In spleen and lung, no OTC mRNA was detectable by Northern blot analysis even after a long exposure time (Fig 6). This result is in agreement with the low activities we detected in these two tissues.
DISCUSSION Wv'e have obtained a correction of the Spf-ash ornithine transcarbamylase deficiency by gene transfer. In the liver, OTC mRNA is 50 % of control values while the enzymatic activity reaches 80% This difference could be due to an enhanced stability of the rat RNA sequence and/or an enhanced stability of the rat enzyme in the mouse environment. The tissue specificity of the expression does not in principle need discussion since first, we have not demonstrated a transcriptionnal regulation of the transoene expression by run-on experiment . Secondly, we have obtained only one transgenic animal and therefore we cannot eliminate the possibility that tissue specificity is determined by integration si-te sequences. Nevertheless, as the transgene expression mimics the endogenous OTC distribution, we may suggest that some regulatory sequences contained in the rat OTC cDNA acting joint'y with the SV 40 promoter may lead to a specificity similar to that of the OTC oene. The fact that the SV 40 core sequence is also present in the 5' flanking region of both murine and human OT.C gene supports this hypothesis (22,23). A similar inheritable tissue-specific expression has been previously reported (24, 25). However, further studies with more transgenic aninals will be required before an effect of inteoration site sequences on the tissue specificity of the expression can be excluded. In conclusion, we have demonstrated the feasibiiity of using the nicroinjection technique to correct a hereditary enzynatic deficiency in mice by the introduction of heterologous cDNA codino
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Nucleic Acids Research for the normal enzyme. The expression of this cDNA controlled by the SV40 early promoter leads to the elimination of the phenotypic traits associated with the OTC mutation and to the restoration of a functional urea cycle.
ACKNOWLEDGEMENTS W,e are grateful to Dr Shore for givino the OTC cDL4A probe. This work was supported by the Institut National de la Sante et de la R.echerche Me'dicale (grant n 86/434) and by the Neckler-Enfants Hialades Hospital Scientific Council.
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Nucleic Acids Research 21. Wilkie, T. M. and Palmiter, R. D. (1987) Mol. Cell. Biol. 7, 1646-1655 22. Veres, G., Craigen, W. J. and Caskey, C. T. (1986) J. Biol. Chem. 261, 7588-7591 23. Hata, A., Tzuzuki, T., Shimada, K., Takiguchi, M., Mori, M. and Matsuda, I. (1986) J. Biochem. 100, 717-725 24. Swanson, L. W., Simmons, D. M., Arriza, J., Hammer, R., R. M. (1986) Brinster, R. L., Rosenfeld, M. G. and Evans, Nature. 317, 363-366
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