A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain

RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor α1 (TRα1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain.

[1]  Judy Lieberman,et al.  RNA interference targeting Fas protects mice from fulminant hepatitis , 2003, Nature Medicine.

[2]  M. Izquierdo,et al.  RNA interference against Hec1 inhibits tumor growth in vivo , 2006, Gene Therapy.

[3]  B. Ramratnam,et al.  Promoter choice affects the potency of HIV-1 specific RNA interference. , 2003, Nucleic acids research.

[4]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[5]  C. Brinckerhoff,et al.  Short hairpin RNA-mediated inhibition of matrix metalloproteinase-1 in MDA-231 cells: effects on matrix destruction and tumor growth. , 2005, Cancer research.

[6]  S. Leoni,et al.  Thyroid Status Affects Rat Liver Regeneration After Partial Hepatectomy by Regulating Cell Cycle and Apoptosis , 2005, Cellular Physiology and Biochemistry.

[7]  D. Scherman,et al.  Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio. , 1995, Human gene therapy.

[8]  B. Demeneix,et al.  Polyethylenimines for in vivo gene delivery. , 2001, Current opinion in molecular therapeutics.

[9]  Phillip D Zamore,et al.  RNAi: nature abhors a double-strand. , 2002, Current opinion in genetics & development.

[10]  S. García-Silva,et al.  The Thyroid Hormone Receptor Is a Suppressor of ras-Mediated Transcription, Proliferation, and Transformation , 2004, Molecular and Cellular Biology.

[11]  F. Chisari,et al.  Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[13]  C. Henderson,et al.  Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS , 2005, Nature Medicine.

[14]  C. Napoli,et al.  Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. , 1990, The Plant cell.

[15]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[16]  M. Mathews,et al.  Interactions between double-stranded RNA regulators and the protein kinase DAI , 1992, Molecular and cellular biology.

[17]  Lemkine Gf,et al.  Polyethylenimines for in vivo gene delivery. , 2001 .

[18]  Seow Theng Ong,et al.  Hybrid cytomegalovirus enhancer-h1 promoter-based plasmid and baculovirus vectors mediate effective RNA interference. , 2005, Human gene therapy.

[19]  Zuoshang Xu,et al.  An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi , 2005, Nucleic acids research.

[20]  W. Forrester,et al.  A DNA vector-based RNAi technology to suppress gene expression in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. Hannon,et al.  Multiple cis-acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P. , 1991, The Journal of biological chemistry.

[22]  C. Baglioni,et al.  Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. , 1979, The Journal of biological chemistry.

[23]  Haibin Xia,et al.  siRNA-mediated gene silencing in vitro and in vivo , 2002, Nature Biotechnology.

[24]  F. Natt,et al.  siRNA relieves chronic neuropathic pain. , 2004, Nucleic acids research.

[25]  N. Hecht,et al.  Translin associated protein X is essential for cellular proliferation , 2004, FEBS letters.

[26]  D. Engelke,et al.  Effective expression of small interfering RNA in human cells , 2002, Nature Biotechnology.

[27]  K. Taira,et al.  Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. , 2003, Nucleic acids research.

[28]  Shu Wang,et al.  CMV enhancer/human PDGF-β promoter for neuron-specific transgene expression , 2004 .

[29]  G. Levi,et al.  Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  G. M. Ledda-Columbano,et al.  Induction of pancreatic acinar cell proliferation by thyroid hormone. , 2005, The Journal of endocrinology.

[31]  Zuoshang Xu,et al.  An enhanced U6 promoter for synthesis of short hairpin RNA. , 2003, Nucleic acids research.

[32]  Stacy L DeRuiter,et al.  RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  D. Strayer,et al.  Paradigms for conditional expression of RNA interference molecules for use against viral targets. , 2005, Methods in enzymology.

[34]  J. Rossi,et al.  Novel Pol II Fusion Promoter Directs Human Immunodeficiency Virus Type 1-Inducible Coexpression of a Short Hairpin RNA and Protein , 2006, Journal of Virology.

[35]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[36]  S. Wang,et al.  CMV enhancer/human PDGF-beta promoter for neuron-specific transgene expression. , 2004, Gene therapy.

[37]  J. Behr,et al.  Lipid‐mediated siRNA delivery down‐regulates exogenous gene expression in the mouse brain at picomolar levels , 2005, The journal of gene medicine.