Research reportTransgene expression of plasmid DNAs directed by viral or neural promoters in the rat brain
Introduction
Several methods, including the use of recombinant RNA or DNA viruses, have been developed to introduce transgenes into the CNS. Viral methods have involved retroviruses 15, 20, adenoviruses (ADV) [41]and herpes simplex virus (HSV1) 9, 22. However, although these replication-incompetent viruses are non-pathogenic, they do result in immune and inflammatory responses against viral proteins others than the transgene product 19, 49, 50.
An alternative approach to the induction of transgene expression within the CNS could be to use plasmids. This has several advantages over the use of recombinant viruses, e.g. plasmids, are only weakly immunogenic [36]. The gene transfer approach using plasmids has been greatly improved [14]and efficient transgene expression has been obtained in muscle [48], heart [1]and liver [26]. A DNA plasmid can be as efficient as adenovirus in inducing transgene expression in regenerating muscle and more efficient than retrovirus in mature muscle [18]. Most of these experiments were carried out with DNA–liposome complex (for review, see [33]). In the brain, most strategies are based on virus as vectors, just few used lipofectin-treated plasmids and suggest that DNA, but RNA also, can be expressed in embryonic brain of Xenopus [27], mouse [42]and rat 13, 28, 35.
In the present study, we have analysed the expression and maintenance of DNA in different regions of the mature rat brain. Naked DNA was used since we observed that in the brain mixtures of polycations and DNA are more toxic than DNA alone. The efficiency and kinetics of transgene (LacZ) expression were compared under the control of three different promoters, a human cytomegalovirus (HCMV) promoter, highly expressed in different cells or the enolase (NSE) or glial fibrillary acidic protein (GFAP) promoters, specific for neurons or astrocytes, respectively. Only the injection of large amounts of naked plasmid DNA (50 or 100 μg) resulted in significant expression of the transgene LacZ. Irrespective of the promoter used, LacZ expression decreased progressively with time over 2 months. Depending on the promoter used (NSE or GFAP), LacZ could be preferentially expressed in neurons or astrocytes, respectively. These data indicate that under appropriate conditions plasmid DNA can be efficiently expressed in the brain.
Section snippets
Plasmid constructs
pCMVLacZ, containing the LacZ/phleomycin fusion gene under the control of the HCMV promoter (pUT535), was obtained from Dr. G. Tiraby [5]. The GFAP promoter, between BglII and HincII from pGFACAT2 (provided by Dr. M. Brenner) [7], was cloned into the KS vector, between the BamHI and SmaI sites. A SpeI-PstI fragment, containing the GFAP promoter, was then inserted into pUT535 between the corresponding sites, replacing the HCMV promoter (pGFAPLacZ). pNSELacZ, containing LacZ under the NSE
Dose–response and comparison of β-gal expression in different regions of the brain
In these experiments, we evaluated the amount of DNA required to obtain consistent β-gal expression and also assessed the possible influence of the site of injection. 1, 5, 10, 50 and 100 μg of naked DNA from pCMVLacZ, the plasmid construct containing LacZ directed by the HCMV promoter (chosen because it has been shown to be a potent ubiquitous promoter of exogenous genes in the CNS [31]) was injected into the thalamus and LacZ expression determined 48 h later by X-gal staining. Previous
Discussion
In the present study, plasmid DNA containing a reporter gene (LacZ) was stereotaxically injected into the rat brain. We studied expression of LacZ under the control of three different promoters, the ubiquitous promoter (HCMV), the neuron-specific promoter (NSE) and the astrocyte-specific (GFAP) promoter. Our results demonstrated that: (1) substantial transgene expression required the injection of large doses (50 μg) of DNA; (2) HCMV and GFAP promoters were more effective than the NSE promoter;
Acknowledgements
We thank Dr. B. Pessac for C8 cells, Dr. G. Tiraby for pUT535, Dr. M. Brenner for pGFAPCAT2, Dr. S. Forss-Petter for pNSELacZ, Dr. J. Honnorat for Hu serum and Dr. I. Akaoka for critical comments. This work was also supported by MRES and DRET. Z.H. was supported by MES (Ministère de l'Enseignement Supérieur d'Algérie) and S.S. by Conicit UCV (Consejo Nacional de Investigation Cientifica y Tecnologica Caracas).
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