Nucleic Acids Research

This Article Abstract Print PDF (180K) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Commercial Re-use Guidelines for Open Access NAR Content Google Scholar Articles by Xia, X. G. Articles by Xu, Z. Search for Related Content PubMed PubMed Citation Articles by Xia, X. G. Articles by Xu, Z. Related Collections Targeted inhibition of gene function Social Bookmarking          What's this?

Nucleic Acids Research, 2003, Vol. 31, No. 17 e100
© 2003 Oxford University Press

An enhanced U6 promoter for synthesis of short hairpin RNA

Xu Gang Xia¹, Hongxia Zhou¹, Hongliu Ding¹, El Bashir Affar⁴, Yang Shi⁴ and Zuoshang Xu^*,1,2,3

¹ Department of Biochemistry and Molecular Pharmacology, ² Department of Cell Biology and ³ Neuroscience Program, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA and ⁴ Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

*To whom correspondence should be addressed. Tel: +1 508 856 3309; Fax: +1 508 856 8390; Email: zuoshang.xu{at}umassmed.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received May 20, 2003; Revised and Accepted June 25, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1^G93A) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
RNA interference (RNAi) can mediate sequence-selective suppression of gene expression in a wide variety of eukaryotes by introducing short RNA duplexes (small interfering RNAs or siRNAs) with sequence homologies to the target gene (1,2). Furthermore, short hairpin RNAs (shRNAs) transcribed in vivo under the control of RNA polymerase III (Pol III) promoters can trigger degradation of corresponding mRNAs similar to siRNAs and inhibit specific gene expression (3–11). Constructs that synthesize shRNA have been incorporated into viral vectors and these vectors can mediate RNAi in culture as well as in vivo (12–16). Therefore, Pol III–shRNA constructs may be developed to mediate long-term silencing of dominant, gain-of-function mutant genes that cause diseases.

In diseases caused by a gain-of-function type of mutation, the mutant is toxic but the wild-type performs important functions. Therefore, the ideal therapy should selectively silence the mutant gene but maintain the wild-type gene expression. Although opinions vary (17–19), many experiments have shown that siRNAs and shRNAs can discriminate between mRNAs that differ at a single nucleotide and selectively degrade the perfectly matched mRNA, while leaving the mRNA with a single nucleotide mismatch unaffected (7,9,12,17,20). The discriminating siRNA or shRNA must include the altered nucleotide in its sequence and, in most instances, the optimal design places the altered nucleotide near or at the middle of the siRNA or shRNA. This limits the sequence selection in designing siRNA or shRNA around the site of mutation. Because the sequence of siRNA or shRNA greatly influences the efficacy of RNAi (18,21), the sequences surrounding the mutation site may not be optimal and could produce poor inhibitors of the mutant gene.

We have designed an shRNA-expressing construct controlled by a Pol III U6 promoter (22) to silence a mutant Cu,Zn superoxide dismutase (SOD1^G93A) allele that causes amyotrophic lateral sclerosis (ALS), a fatal degenerative motor neuron disease (23). While testing the efficacy of this shRNA, we found it selectively inhibited the expression of a mutant SOD1^G93A but did not affect SOD1^WT (24). However, the efficacy of RNAi produced by this construct was relatively modest, which might affect the ultimate therapeutic efficacy. One way to overcome this problem was to increase the dose of the shRNA by enhancing the Pol III promoter activity. We realized that some snRNAs are synthesized by Pol II while others are synthesized by Pol III, and they share enhancer elements (25–30). Hence, a Pol II enhancer might be able to enhance the Pol III transcription. We tested this by placing the enhancer from the cytomegalovirus (CMV) promoter near the U6 promoter and demonstrated that this enhanced the U6 promoter activity, increased the shRNA synthesis and strengthened the silencing of the target gene. This enhanced promoter may be broadly useful in similar situations in targeting other disease-associated mutants.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plasmid construction
The SOD1^G93AGFP fusion plasmid was constructed as described before (24). Briefly, mutant human SOD1^G93A cDNA was PCR cloned between the PmlI and PstI sites of pCMV/myc/mito/GFP (Invitrogen). This cloning step deleted the mitochondrial targeting sequence. U6G93Ahp was constructed as described (6). Similarly, U6misG93A was created using the sequence GACAAAGCTGCTGTATCGGCT (sense strand), which contains five mismatched nucleotides (bold) against the SOD1^G93A mutant. The CMV enhancer was PCR cloned from the pDsRed2-N1 vector (nucleotides 1–484; Clontech) and inserted either upstream between KpnI and NheI or downstream between NotI and SacI sites of U6G93Ahp.

Cell culture and transfection
Human embryonic kidney cell line 293 was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Twenty-four hours before transfection, cells (70–90% confluency) were detached by trituration, transferred to 6-well plates and cultured in 10% FBS-containing medium without antibiotics. The cells were transfected with 4 µg of the target vector SOD1^G93AGFP and 8 µg of each of the hairpin vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The transfection efficiency was 95% in all experiments. After 24 h, the culture medium was changed to DMEM supplemented with 10% FBS and antibiotics. At 40 h after transfection, the cells were harvested and quickly frozen in liquid nitrogen.

Measurement of GFP fluorescence intensity
The harvested cells were lysed in ice-cold reporter lysis buffer (Promega) containing protease inhibitors (complete, EDTA-free, 1 tablet/10 ml buffer; Roche Molecular Biochemicals). The lysate was cleared by centrifugation at 16000 g and 4°C for 10 min. The total protein in the cleared lysate was measured using the BCA assay (Pierce, Rockville, IL). The total protein concentration in each sample was adjusted to 0.5 mg/ml with the reporter buffer. Fluorescence of GFP in 140 µl of sample was measured by fluorescence spectroscopy (Photon Technology International) with excitation at 460 nm and recording from 480 to 600 nm. The spectrum peak was detected at 502 nm, representing the fluorescence intensity of GFP. Fluorescence in the untransfected lysate was measured as background and subtracted from measurements of the transfected lysates.

Western blots
Twenty micrograms of total protein were resolved on a 12% SDS–PAGE gel and transferred onto GeneScreen Plus membrane (Perkin Elmer). The membrane was incubated sequentially with a sheep anti-SOD1 (BioDesign) and horseradish peroxidase-labeled goat anti-sheep IgG (Amersham). The protein bands were visualized using SuperSignal kit (Pierce) and Kodak Digital Image Station 440CF.

Northern blot analysis
Cellular RNA was isolated with TRI reagent (Sigma). Twenty micrograms of total RNA were fractionated on a 15% polyacrylamide gel and transferred to Hybond^TM-N⁺ membrane (Amersham). The membrane was probed with ³²P-labeled synthetic RNA oligonucleotide complementary to the antisense strand of the G93A hairpin.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The shRNA against SOD1^G93A (G93Ahp) contains a stem that is homologous to SOD1^G93A mRNA but has a mismatched nucleotide with SOD1^WT at the middle of the stem (Fig. 1A). When transfected into cultured cells, this shRNA selectively inhibited the expression of SOD1^G93A but did not affect the expression of SOD1^WT (24) (also see below). However, the RNAi efficacy of this shRNA was relatively poor (see below). We therefore sought to increase the potency of this shRNA by increasing its expression. We modified the U6 promoter by placing the enhancer from the CMV immediate-early promoter near the U6 promoter, either upstream or downstream from U6G93Ahp and in either forward or reverse orientation (Fig. 1B).

View larger version (29K): [in this window] [in a new window]   Figure 1. Design of hairpin constructs against mutant SOD1G93A. (A) Nucleic acid sequence surrounding the mutation site of SOD1G93A. Notice the G (bold) in the antisense strand of the hairpin that matches with the C (bold) in SOD1G93A but mismatches with a G in SOD1WT. (B) Variations of the U6 promoter. U6G93Ahp, authentic U6 promoter with G93A hairpin; EN-U6G93Ahp, forward CMV enhancer placed 5' of the U6 promoter; REN-U6G93Ahp, reverse CMV enhancer placed 5' of the U6 promoter; U6G93Ahp-EN, forward CMV enhancer placed 3' of U6G93Ahp; U6G93Ahp-REN, reverse CMV enhancer placed 3' of U6G93Ahp; EN-U6G93Ahp, forward CMV enhancer placed 5' of the crippled U6 promoter with DSE deletion.

 
We co-transfected each of the seven constructs containing various combinations of U6 promoter, G93Ahp and CMV enhancer, with a target construct that encoded a SOD1^G93A and GFP fusion protein (SOD1^G93AGFP), into human 293 cells. Northern blot analysis demonstrated that addition of the CMV enhancer near U6G93Ahp in all four configurations (Fig. 1) increased the expression of G93Ahp (Fig. 2). Deletion of the distal sequence element (DSE), an obligatory component of the U6 promoter (25–28), abolished expression of G93Ahp even in the presence of the enhancer (Fig. 2). Quantification of SOD1^G93AGFP expression by GFP fluorescence using a fluorometer (31) showed that, compared with SOD1^G93AGFP alone transfection (Fig. 3A and B, 1), G93Ahp produced by the unmodified U6 promoter (U6G93Ahp) inhibited SOD1^G93AGFP expression modestly (Fig. 3A and B, 2). Attaching the CMV enhancer in all four configurations to U6G93Ahp (Fig. 1B) enhanced the inhibition of SOD1^G93AGFP expression to a similar degree (Fig. 3A and B, 3–6). Deletion of the DSE abolished the inhibition of SOD1^G93AGFP expression (Fig. 3A and B, 7). Finally, U6 promoter directed synthesis of mismatched shRNA did not show any inhibitory activity towards the target gene (Fig. 3A and B, 8).

View larger version (90K): [in this window] [in a new window]   Figure 2. Northern blot detecting the G93Ahp transcripts. Total RNA was extracted from human 293 cells doubly transfected with SOD1G93AGFP and various U6G93Ahp constructs (Fig. 1B). G93Ahp was detected using a 32P-labeled 21 nt RNA probe complementary to the antisense strand of the hairpin stem. Note the enhanced expression of the shRNA in cells transfected with the U6G93Ahp constructs containing the CMV enhancer.

 

View larger version (29K): [in this window] [in a new window]   Figure 3. CMV enhancer increases the inhibition of target gene expression. (A) Fluorometer measurement of GFP fluorescence in lysates from the 293 cells transfected with SOD1G93AGFP and various U6G93Ahp constructs (Fig. 1B). (B) Average peak GFP fluorescence intensity from the six independent experiments shown in (A). The numbers on the x-axis correspond to the numbers in the legend to (A). Error bars indicate SEM. (C) Immunoblot of SOD1. The lane numbers correspond to the numbers in the legend to (A).

 
Western blot using a polyclonal anti-SOD1 antibody confirmed the above finding and, furthermore, showed that the enhanced synthesis of G93Ahp only inhibited SOD1^G93AGFP expression but did not affect the endogenous human SOD1 levels (Fig. 3C), indicating that the high levels of G93Ahp expression do not affect the specificity of G93Ahp for the mutant SOD1^G93A.

Our results demonstrate that the CMV enhancer can enhance U6 promoter activity and increase the production of shRNA. This modified promoter may be useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region and, therefore, could be used for selective inhibition of mutant gene expression in vitro and in vivo and to develop therapies for diseases caused by dominant, gain-of-function gene mutations.

	ACKNOWLEDGEMENTS

 
We thank Dr Guiliang Tang for teaching us northern blotting, Drs Phillip Zamore and Theresa Zhang for critically reading the manuscript and members of the Xu laboratory for advice and support. This work was supported by grants from the ALS Association, NINDS (RO1 NS41739 and NS35750) and The Robert Pachard Center for ALS Research at Johns Hopkins to Z.S.X. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the NINDS.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1. Elbashir,S.M., Harborth,J., Lendeckel,W., Yalcin,A., Weber,K. and Tuschl,T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.[CrossRef][Medline]

2. Caplen,N.J., Parrish,S., Imani,F., Fire,A. and Morgan,R.A. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl Acad. Sci. USA, 98, 9742–9747.[Abstract/Free Full Text]

3. Paul,C.P., Good,P.D., Winer,I. and Engelke,D.R. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol., 20, 505–508.[CrossRef][Web of Science][Medline]

4. Lee,N.S., Dohjima,T., Bauer,G., Li,H., Li,M.J., Ehsani,A., Salvaterra,P. and Rossi,J. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol., 20, 500–505.[Web of Science][Medline]

5. Paddison,P.J., Caudy,A.A., Bernstein,E., Hannon,G.J. and Conklin,D.S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev., 16, 948–958.[Abstract/Free Full Text]

6. Sui,G., Soohoo,C., Affar, el B., Gay,F., Forrester,W.C. and Shi,Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl Acad. Sci. USA, 99, 5515–5520.[Abstract/Free Full Text]

7. Yu,J.Y., DeRuiter,S.L. and Turner,D.L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl Acad. Sci. USA, 99, 6047–6052.[Abstract/Free Full Text]

8. McManus,M.T., Petersen,C.P., Haines,B.B., Chen,J. and Sharp,P.A. (2002) Gene silencing using micro-RNA designed hairpins. RNA, 8, 842–850.[Abstract]

9. Brummelkamp,T.R., Bernards,R. and Agami,R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553.[Abstract/Free Full Text]

10. Miyagishi,M. and Taira,K. (2002) U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol., 20, 497–500.[CrossRef][Web of Science][Medline]

11. Jacque,J.M., Triques,K. and Stevenson,M. (2002) Modulation of HIV-1 replication by RNA interference. Nature, 418, 435–438.[CrossRef][Medline]

12. Brummelkamp,T., Bernards,R. and Agami,R. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell, 2, 243.[CrossRef][Web of Science][Medline]

13. Hasuwa,H., Kaseda,K., Einarsdottir,T. and Okabe,M. (2002) Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett., 532, 227–230.[CrossRef][Web of Science][Medline]

14. Xia,H., Mao,Q., Paulson,H.L. and Davidson,B.L. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol., 20, 1006–1010.[CrossRef][Web of Science][Medline]

15. Tiscornia,G., Singer,O., Ikawa,M. and Verma,I.M. (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl Acad. Sci. USA, 100, 1844–1848.[Abstract/Free Full Text]

16. Rubinson,D.A., Dillon,C.P., Kwiatkowski,A.V., Sievers,C., Yang,L., Kopinja,J., Zhang,M., McManus,M.T., Gertler,F.B., Scott,M.L. et al. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nature Genet., 33, 401–406.[CrossRef][Web of Science][Medline]

17. Boutla,A., Delidakis,C., Livadaras,I., Tsagris,M. and Tabler,M. (2001) Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila. Curr. Biol., 11, 1776–1780.[CrossRef][Web of Science][Medline]

18. Holen,T., Amarzguioui,M., Wiiger,M.T., Babaie,E. and Prydz,H. (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res., 30, 1757–1766.[Abstract/Free Full Text]

19. Zeng,Y. and Cullen,B.R. (2003) Sequence requirements for micro RNA processing and function in human cells. RNA, 9, 112–123.[Abstract/Free Full Text]

20. Elbashir,S.M., Martinez,J., Patkaniowska,A., Lendeckel,W. and Tuschl,T. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J., 20, 6877–6888.[CrossRef][Web of Science][Medline]

21. Elbashir,S.M., Harborth,J., Weber,K. and Tuschl,T. (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 26, 199–213.[CrossRef][Web of Science][Medline]

22. Kunkel,G.R., Maser,R.L., Calvet,J.P. and Pederson,T. (1986) U6 small nuclear RNA is transcribed by RNA polymerase III. Proc. Natl Acad. Sci. USA, 83, 8575–8579.[Abstract/Free Full Text]

23. Cleveland,D.W. and Rothstein,J.D. (2001) From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nature Rev. Neurosci., 2, 806–819.[CrossRef][Web of Science][Medline]

24. Ding,H.L., Schwarz,D.S., Keene,A., Affar,E.B., Fenton,L., Xia,X.G., Shi,Y., Zamore,P.D. and Xu,Z.S. (2003) Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Aging Cell, 2, 209–217.[CrossRef][Web of Science][Medline]

25. Bark,C., Weller,P., Zabielski,J., Janson,L. and Pettersson,U. (1987) A distant enhancer element is required for polymerase III transcription of a U6 RNA gene. Nature, 328, 356–359.[CrossRef][Medline]

26. Carbon,P., Murgo,S., Ebel,J.P., Krol,A., Tebb,G. and Mattaj,L.W. (1987) A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II. Cell, 51, 71–79.[CrossRef][Web of Science][Medline]

27. Kunkel,G.R. and Pederson,T. (1988) Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Genes Dev., 2, 196–204.[Abstract/Free Full Text]

28. Das,G., Henning,D., Wright,D. and Reddy,R. (1988) Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. EMBO J., 7, 503–512.[Web of Science][Medline]

29. Lobo,S.M. and Hernandez,N. (1989) A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell, 58, 55–67.[CrossRef][Web of Science][Medline]

30. Mattaj,I.W., Dathan,N.A., Parry,H.D., Carbon,P. and Krol,A. (1988) Changing the RNA polymerase specificity of U snRNA gene promoters. Cell, 55, 435–442.[CrossRef][Web of Science][Medline]

31. Chiu,Y.-L. and Rana,T.M. (2002) RNAi in human cells: basic structural and functional features of small interfering RNA. Mol. Cell, 10, 549–561.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				S. Dupraz, D. Grassi, M. E. Bernis, L. Sosa, M. Bisbal, L. Gastaldi, I. Jausoro, A. Caceres, K. H. Pfenninger, and S. Quiroga The TC10-Exo70 Complex Is Essential for Membrane Expansion and Axonal Specification in Developing Neurons J. Neurosci., October 21, 2009; 29(42): 13292 - 13301. [Abstract] [Full Text] [PDF]

				A.-M. Anesti, P. J. Peeters, I. Royaux, and R. S. Coffin Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus Nucleic Acids Res., August 1, 2008; 36(14): e86 - e86. [Abstract] [Full Text] [PDF]

				Z. Hassani, J.-C. Francois, G. Alfama, G. M. Dubois, M. Paris, C. Giovannangeli, and B. A. Demeneix A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain Nucleic Acids Res., May 14, 2007; 35(9): e65 - e65. [Abstract] [Full Text] [PDF]

				C. Haffner, U. Dettmer, T. Weiler, and C. Haass The Nicastrin-like Protein Nicalin Regulates Assembly and Stability of the Nicalin-Nodal Modulator (NOMO) Membrane Protein Complex J. Biol. Chem., April 6, 2007; 282(14): 10632 - 10638. [Abstract] [Full Text] [PDF]

				T. M. Wise-Draper, H. V. Allen, E. E. Jones, K. B. Habash, H. Matsuo, and S. I. Wells Apoptosis Inhibition by the Human DEK Oncoprotein Involves Interference with p53 Functions Mol. Cell. Biol., October 15, 2006; 26(20): 7506 - 7519. [Abstract] [Full Text] [PDF]

				H. Zhou, X. G. Xia, and Z. Xu An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi Nucleic Acids Res., April 1, 2005; 33(6): e62 - e62. [Abstract] [Full Text] [PDF]

				I Bantounas, L A Phylactou, and J B Uney RNA interference and the use of small interfering RNA to study gene function in mammalian systems J. Mol. Endocrinol., December 1, 2004; 33(3): 545 - 557. [Abstract] [Full Text] [PDF]

				F. Schlachetzki, Y. Zhang, R. J. Boado, and W. M. Pardridge Gene therapy of the brain: The trans-vascular approach Neurology, April 27, 2004; 62(8): 1275 - 1281. [Abstract] [Full Text] [PDF]

This Article Abstract Print PDF (180K) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Commercial Re-use Guidelines for Open Access NAR Content Google Scholar Articles by Xia, X. G. Articles by Xu, Z. Search for Related Content PubMed PubMed Citation Articles by Xia, X. G. Articles by Xu, Z. Related Collections Targeted inhibition of gene function Social Bookmarking          What's this?

Online ISSN 1362-4962 - Print ISSN 0305-1048

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics