Genes In Mammalian Cells Research Articles

1059. Development of a Novel Regulatory System Allowing Doxycycline-Controlled Suppression of Target Genes in Mammalian Cells

Top of pageAbstract The efficient and specific suppression of target genes by RNA interference (RNAi) is a remarkable new approach that may find applications in human therapy whenever genes involved in the respective pathology have to be inhibited. Clinical applications will require a regulatory system that allows control of RNAi by administration of a small inducer molecule. The treatment could then be adapted to the needs of the patient and, should complications arise, the therapy could be stopped or interrupted. RNAi based gene suppression can be induced in target cells by short hairpin RNAs (shRNAs) that are expressed under the control of RNA polymerase III promoters in order to avoid disturbing poly A sequences. To control RNAi by inducible and reversible expression of shRNAs, we developed a novel regulatory system. This system is based on a novel tetracycline-dependent transactivator capable of inducing transcription of shRNAs from a minimal RNA polymerase III promoter in the presence of doxycycline. As a proof-of-principle, tetracycline-controlled RNA interference was used to regulate the expression of GFP in HEK 293T cells stably expressing this transgene. RNA interference was induced by administration of doxycycline yielding a 90% reduction of GFP expression. The effect of doxycycline on the expression of GFP was dose- and time-dependent. In particular, the down-regulation of GFP was reversible after withdrawal of doxycycline, as observed by reappearance of GFP fluorescence.

381. Methods for Optimizing Lentiviral Vector-Mediated Gene Suppression by RNA Interference

Short interfering (si)RNA methods have the potential for specific suppression of target genes, but finding the optimal siRNA target sequence and evaluating the persistence of gene knockdown are challenges which hinder progress. We have developed a strategy for vector-based screening of multiple siRNA sequences against target genes in mammalian cells. The steps are as follows: 1) creation of a fusion construct of the gene of interest (GOI) and a fluorescent reporter such as GFP at either the N-or C-terminus, 2) stable expression of the GOI-GFP fusion in a mammalian cell line, 3) transfection of vector plasmids encoding templates for short-hairpin (sh)RNAs, under the transcriptional control of a human RNA polymerase III (H1) promoter, targeted against several GOI sequences in the stable cell line, 4) quantification of the GOI knockdown by flow cytometric analysis for diminished GFP fluorescence. Quantification of expression by this method permits not only the selection of the most suppressive sequences but also those capable of resulting in intermediate levels of suppresion and hence intermediate phenotypic outcomes. In a modification of step 3, the shRNA expression cassettes are subcloned into a lentiviral vector system permitting transduction of stably modified cells thereby enabling the evaluation of persistence of the suppressive effect after multiple subcultures.

1061. An Ecdysone Receptor-Based Gene Switch for Precisely and Variably Regulated Expression of Genes in Mammalian Cells

Top of pageAbstract Different versions of an ecdysone receptor (EcR)-based gene switch that can be regulated by synthetic non-steroid ligands have been developed and reported previously. The most active ligands that are used for regulating these switch systems are the diacylhydrazine class of compounds that exhibit very favorable toxicological properties in the mammalian cells. The gene switch is designed in a two-hybrid format and consists of two nuclear receptors that heterodimerize to form the transcriptional activator. The receptor that binds the activating ligand is the DEF domain of EcR that is fused to a DNA binding domain (DBD) such as Gal4 (1|[ndash]|147 a.a.). The heterodimerizing partner is the EF domain of a chimeric RXR that is fused to a transcriptional activation domain such as VP16. Ligand binding to the EcR enhances its interaction with its RXR partner and activates transcription from a responsive promoter that contains the binding sites for the DBD-EcR(DEF). The gene of interest that is inserted downstream of the responsive promoter can be regulated in a dose-dependent manner by the ligand. A repertoire of inducible promoters have been incorporated into different versions of the switch. Data will be presented in which the gene switch provides very tight but moderate expression levels for genes that require stringent regulation. At the other end of this spectrum are the inducible promoters allowing slightly leaky but very high expression levels for applications where the absolute expression levels are more important. The switch system was originally designed as a three-vector system in which the two receptor components and the inducible expression elements were on three separate vectors. Presently, the gene switch has been assembled into a single vector system that accommodates all the three components of the switch as a single all-in-one cassette. In the initial all-in-one vector, the two receptor components were expressed from two different constitutive promoters. In the later versions, the receptors are expressed from a single constitutive promoter as a dicistronic message with an IRES sequence in the middle. Modifications in the switch design resulted in an all-in-one cassette that is small enough to be accommodated into viral vectors.

Histone Dynamics on the Interleukin-2 Gene in Response to T-Cell Activation

Several models have been proposed for the mechanism of chromatin remodelling across the promoters of inducible genes in mammalian cells. The most commonly held model is one of cooccupation where histone proteins are modified by acetylation or phosphorylation and nucleosomes are remodelled, allowing the assembly of transcription factor complexes. Using chromatin immunoprecipitation, we observed an apparent decrease of histone acetylation and phosphorylation signals at the proximal promoter region of the inducible interleukin-2 and granulocyte-macrophage colony-stimulating factor genes in response to T-cell activation. We showed that this apparent decrease was due to a loss of histone H3 and H4 proteins corresponding to a decrease in nucleosome occupation of the promoter. This histone loss is reversible; it is dependent on the continual presence of appropriate activating signals and transcription factors and is not dependent on the acetylation status of the histone proteins. These data show for the first time that histone proteins are lost from a mammalian promoter upon activation of transcription and support a model of activation-dependent disassembly and reassembly of nucleosomes.

The use of small interfering RNA to elucidate the activity and function of ion channel genes in an intact tissue

Small interfering RNA (siRNA) directs the targeted destruction of mRNA encoding a specific protein, in a process known as RNA interference (RNAi). This stops translation of the targeted mRNA into protein, effectively silencing the gene. RNAi is a recent discovery, identified in mammalian cells in 2001, but it has rapidly advanced into a practical technique and is being used increasingly to investigate mammalian gene function. Tools are available to induce RNAi in cell lines, intact tissue preparations and even in vivo. Depending on the method used, loss of gene expression may be transient or sustained, enabling a wide range of functions to be investigated. RNAi therefore offers a powerful technique that can be used to produce targeted knockout of ion channel genes in mammalian cells. Its applications potentially include identification of ion channel function in health and disease, identification of novel channel genes and drug target validation. This paper outlines our current understanding of siRNA and the experimental requirements for producing efficient RNAi and gene silencing. Effective RNAi requires an appropriate siRNA sequence to be designed and an efficient method for delivering the siRNA to the cells of interest. Since not all potential siRNA sequences are effective, it is also important to verify the loss of gene expression by measuring the level of channel protein remaining. Limitations of the methods available for delivering siRNA are one of the main obstacles to producing efficient RNAi, especially in intact tissue preparations. Here we describe an in vitro method for targeted RNAi against the TASK-1 potassium channel gene in an isolated vascular preparation, using a DNA construct to direct the expression of siRNA, along with a non-viral method for transfecting cells within the vessel. Successful silencing of the TASK-1 gene is verified by immunostaining with an antibody directed against the TASK-1 protein.

Silencing of CXCR4 Blocks Breast Cancer Metastasis

Abstract RNA interference technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. For the first time in cancer research, we show that direct injection of a pool of naked small interfering RNA (siRNA) duplexes can prevent tumorigenesis in an animal model, suggesting a novel preventive and therapeutic strategy for cancer management. As a model system, we used siRNA duplexes of CXCR4 to block breast cancer metastasis. Here, we show that blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis.

Radiation responsive transcriptional regulation of fra-2 gene in mammalian cells.

Radiation responsive transcriptional regulation of fra-2 gene in mammalian cells.

Frequency and Distribution of AP-1 Sites in the Human Genome

The AP-1-binding sequences are promoter/enhancer elements that play an essential role in the induction of many genes in mammalian cells; however, the number of genes containing AP-1 sites remains unknown. In order to better address the overall effect of AP-1 on expression of genes encoded by the entire genome, a genome-wide analysis of the frequency and distribution of AP-1 sites would be useful; yet to date, no such analysis of AP-1 sites or any other promoter/enhancer elements has been performed. We present here our study of the consensus AP-1 site and two single-bp variants showing that the frequency of AP-1 sites in promoter regions is significantly lower than their average rate of occurrence in the whole genomic sequence, as well as the frequency of a random heptanucleotide suggesting that nature has selected for a decrease in the frequency of AP-1 sites in the regulatory regions of genes. In addition, genes containing multiple AP-1 sites are more prevalent than those containing only one copy of an AP-1 site, which again may have evolved to allow for greater signal amplification or integration in the regulation of AP-1 target genes. However, the number of AP-1-regulated genes identified in various studies is far smaller than the number of genes containing potential AP-1 sites, indicating that not all AP-1 sites are activated in a given cell under a given condition, and is consistent with the prediction by others that cellular context determines which AP-1 sites are targeted by AP-1.

Using siRNA Technique to Generate Transgenic Animals with Spatiotemporal and Conditional Gene Knockdown

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.

Hitting the genetic off switch.

I n 1996 Worth magazine proclaimed that Isis Pharmaceuticals could become the next Microsoft, a prediction that turned out to be a particularly egregious example of hyperbole run amok. To be sure, Isis remains a leader in the gene-blocking technology called antisense. But the road to successful treatments for cancer and other diseases has been littered with disappointments. During the past few years, a new genesilencing technology has emerged that may be poised to fulfill the promise that was once trumpeted for antisense. “I’ve been writing in grants for 25 years that during the next five years I’m going to test this process or that process to see if I can do gene inactivation studies in mammalian cells in culture. And I did them, and they were so awkward and so complicated that you just couldn’t apply them generally,” says Phillip A. Sharp, director of the McGovern Institute for Brain Research at the Massachusetts Institute of Technology. “Lo and behold, all of the time right there in front of me was a process that I could have used.” Sharp, a co-winner of the 1993 Nobel Prize in Physiology or Medicine, was referring to a series of relatively recent discoveries that cells have a mechanism, dubbed RNA interference (RNAi), which blocks gene expression. It prevents RNA transcripts of genes from giving rise to the proteins those genes encode. This natural method of gene silencing comes into play, for example, when viruses try to commandeer a cell’s protein-making machinery to produce viral proteins. A milestone arrived in 1998, when Andrew Z. Fire, now at the Stanford University School of Medicine, and Craig C. Mello of the University of Massachusetts Medical School identified in worms double-stranded RNAs that acted as the switch to turn off genes in RNAi. And in 2001 Thomas Tuschl, now at the Rockefeller University, found that an abbreviated version of double-stranded RNAs— short interfering RNAs (siRNAs)—could shut off genes in mammalian cells. The number of research papers on RNAi has mushroomed from a dozen-plus in 1998 to multiple hundreds last year. Even if the promise for therapeutics never materializes, it is quite likely that some of the seminal discoveries will garner Nobel Prizes. “This has touched everything we do in biological science, from plants to man,” Sharp notes. [See “Censors of the Genome,” by Nelson C. Lau and David P. Bartel; Scientific American, August 2003.] The excitement about siRNAs as drugs relates to how they differ in critical ways from antisense therapeutics. At first glance, siRNAs seem very similar to antisense. An antisense drug consists of an artificially synthesized chain of nucleotides, or genetic building blocks, that binds to a messenger RNA containing a complementary sequence. This binding blocks gene expression. An siRNA also silences genes— and it even uses a complementary RNA, or antisense, strand to do so. Once inside a cell, an siRNA attaches to an aggregate of proteins called an RNA-induced silencing complex (RISC), which retains only the antisense strand. The siRNA-bearing RISC then binds to the targeted messenger RNA and degrades it or prevents it from functioning [see box on page 100]. Unlike the antisense drugs that have been under development for the past 15 years, siRNAs do not disrupt only a single messenger RNA. They act as catalysts, doing the same job over and over, one explanation for their apparent potency. “They are 100to 1,000-fold more effective than antisense,” says Judy Lieberman, a senior investigator at the CBR Institute for Biomedical Research in Boston and one of the first researchers to show the therapeutic potential of the technique in animals. Already almost 100 companies are

Silencing of hdm2 oncogene by siRNA inhibits p53-dependent human breast cancer.

RNA interference technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. Here our results showed that hdm2-siRNA silenced its target mRNA specifically and effectively in human breast cancer cells, reduced tumor cell proliferation and induced apoptotic cell death. Other molecular features modified by hdm2-siRNA included decreased Bcl-2, NF-kappaB, survivin, Ras and Raf levels, elevated p53, p21, BRCA1, Bax, and caspase levels as well as altered expression of other genes. hdm2-siRNA also caused cell cycle arrest at G1 phases with reduction in cyclin and Cdk proteins. In addition, hdm2-siRNA displayed in vivo antitumor activity and increased therapeutic effectiveness of mitomycin in MCF-7 xenografts. Thus, hdm2-siRNA may be a promising gene-specific drug for the treatment of human breast cancer and other tumors.

Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells

The expression of recombinant proteins following transduction of CHO cells with recombinant baculoviruses containing a mammalian expression cassette with the CMV-promoter is enhanced by the addition of trichostatin A (TSA), a specific histone deacetylase inhibitor. To further investigate the effect of TSA treatment on protein production following BacMam transduction, viruses containing various viral promoters (SV40, CMV, and RSV) and one cellular promoter (human ubiquitin C) were compared with regard to expression level of a gfp-luciferase fusion protein following transduction of CHO, COS-1, and HEK293 cells. The overall effect on expression appears to be cell specific, indicating that different mechanisms are active within different cell lines. Further, COS cells transfected with naked viral DNA, plasmids, and baculovirus particles were compared in regard to TSA treatment. The increase in reporter gene expression observed following BacMam transduction and TSA treatment were greater than those for transfection of either naked viral DNA or plasmid DNA.

Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells

Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently.

Poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNA from a pol II promoter

Most short hairpin RNA (shRNA)-expressing vectors use RNA polymerase III promoters, as expression of shRNAs from RNA polymerase II promoters is not well understood, due to the lack of defined transcription initiation sites and functional localization of the transcription termination signal. Here we describe a modified cytomegavirus (CMV) promoter, an RNA polymerase II promoter, to express shRNAs with only four overhangs at the 5′ end. The expression of shRNAs from the modified CMV promoter was terminated by the transcription termination, the polyadenylation, or the poly(U) termination signal. These results demonstrated that the poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNAs from the CMV promoter and result in similar efficacies in inhibiting endogenous target genes in mammalian cells.

Differential activation of vascular genes by hypoxia in primary endothelial cells

Changes in the local environment, such as reduced oxygen tension (hypoxia), elicit transcriptional activation of a variety of genes in mammalian cells. Here we have analyzed the effect of hypoxia in different vascular endothelial cells (ECs) with emphasis on hypoxia-regulated transcription factors and genes of importance for blood vessel dynamics. While hypoxia induced the transcription factor hypoxia-inducible factor-1α (HIF-1α) in all endothelial cells tested, the closely related HIF-2α protein was markedly induced in microvascular/capillary endothelial cells, but only weakly or not at all in artery and vein endothelial cells. Furthermore, microvascular/capillary endothelial cells responded to hypoxia with increased number of transcripts encoding vascular endothelial growth factor-A (VEGF-A), VEGF receptor-2, the angiopoietin receptor Tie2, platelet-derived growth factor-B (PDGF-B), and inducible nitric oxide synthase (iNOS). In vein endothelial cells, hypoxia instead increased transcripts encoding lymphatic vascular components VEGF-C, -D, and VEGF receptor-3. Finally, reduced VEGF receptor levels and phosphorylation indicated establishment of a functional autocrine VEGF-A loop in hypoxic endothelial cells. Our results show that endothelial cells, derived from different vascular beds, mount different transcriptional responses to changes in oxygen tension.

Modification of the erythroid transcription factor GATA-1 by SUMO-1.

The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein-protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been shown to regulate important transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We show that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger endogenous globin expression in Xenopus explants. These observations open interesting questions about the biological role of this posttranslational modification of GATA-1.

High-Throughput Gateway Bicistronic Retroviral Vectors for Stable Expression in Mammalian Cells: Exploring the Biologic Effects of STAT5 Overexpression

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 5' of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning.

Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells.

Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. However, the intracellular behavior and properties of the enzyme are not well characterized. Specifically, information on the intracellular kinetics and stability of luciferase activity is necessary for real-time luminescence counts from live cells to be quantitatively meaningful. Here, we report a dynamic analysis of luciferase activity in the context of living mammalian cells. We have determined the relative light units measured in living cells to be proportional to that found in cell lysate. We have also calculated the K(m) of luciferase in living cells to be approximately 1 mM, a value much higher than the 10 microM found for pure enzyme in vitro. In addition, a 2-hour half-life of luciferase activity in live cells was measured in real time. Finally, we have modeled luciferase activity in live cells for the purposes of understanding and translating the luciferase signal into a more effective metric of gene expression and cell behavior.

178. Site-Specific Base Alteration in the Promoter Region of the Human Gamma Globin Gene by Single-Stranded Oligonucleotides for Gene Therapy of Sickle Cell Disease

Top of pageAbstract The human –globin chain has shown greater capacities for inhibiting the polymerization of sickle (S) hemoglobin (Hb) than the –globin chain itself. Moreover, increasing the synthesis of fetal (F) Hb in adults by specific pharmacologic agents has been developed as an alternative strategy to ameliorate sickle cell anemia and –thalassemia. Unfortunately, their clinical application is hampered by their considerable toxicity. Some naturally occurring point mutations in the promoters of A– and G– globin genes are associated with increased HbF in adult erythroid cells. In particular, the C to G mutation at –202 in the G– gene promoter increases the level of HbF up to 15–25% of total hemoglobin, which is sufficient for ameliorating the clinical manifestations of sickle cell anemia. Thus, reactivation of –globin gene expression by targeted mutagenesis of specific sites provides a novel strategy for gene therapy of sickle cell disease. Short (45–100 nt) single-stranded oligonucleotides (SSOs) have been shown to mediate site-specific alteration of single base pairs in both episomal and chromosomal target genes in mammalian cells. The exchange reaction can result in either repair of a lesion or creation of a mutation and is mediated through endogenous DNA repair pathway(s). The aim of this study was to determine if SSO directed DNA repair could introduce specific single base changes in the G– gene promoter region. Forty-five mer SSOs were designed homologous to the G– globin promoter target site –202 except for the middle base which was designed to induce a C to G mutation. This single base alteration at nt –202 also changes the wild-type (wt) Ban II restriction endonuclease site to an Ehe I site, providing a restriction fragment length polymorphism (RFLP) for detection of the desired C to G change. To increase resistance to intracellular nucleases, the SSOs were synthesized with either amine or phosphorothioate linkages at both ends. At various times following transfection of human cell lines with the modified SSOs, genomic DNA was isolated from transfected and control cells and subjected to Ban II digestion. After agarose gel separation of the digested DNA, the genomic DNA from 2.3–3 kb was excised, purified and used as the template for PCR amplification of the G– promoter region spanning the target site. Only cells treated with the SSOs exhibited the desired site-specific C to G change detected by PCR-RFLP. DNA sequencing confirmed that this was the only nt change introduced in the region targeted by the SSO. Moreover, we developed a sensitive and reliable detection system which is suitable for detection of >0.1% point mutations in a wt background. Finally, our methodology for detection indicates that the C to G change was not a PCR artifact. In particular, electrophoretic separation of the digested genomic DNA was done prior to the PCR amplification for the RFLP analysis. In conclusion, 45 mer end-modified SSOs can be used to generate site-specific point mutations in the G– globin gene promoter region to specifically increase HbF expression in adult erythroid cells. This novel strategy provides a gene therapy approach for sickle cell disease and other hemoglobinopathies, independent of correcting the primary genetic lesion in the Hb gene.

994. Gene Knockdown in Mice by Using Lentiviral Vectors Expressing Small Interfering RNA

Top of pageAbstract Many approaches have been described recently for generating loss-of-function phenotypes in eukaryotic systems using RNAi. Use of RNAi by means of long dsRNAs (>50bp) is limited in mammalian cells due to the induction of a nonspecific inhibitory response resulting from the interferon pathway. This limitation was overcome by the finding that small interfering RNAs (siRNAs) can specifically silence genes in mammalian cells without activating the nonspecific interferon pathway. We have designed lentiviral vectors expressing siRNAs to knock down the expression of specific genes. We have previously shown that a lentiviral vector capable of expressing siRNA specific for GFP (siGFP) was able to silence GFP expression in TgGFP mice upon transduction of fertilized mouse embryos. We have found that transgenesis of mouse embryos can be achieved both by zona pellucida removal prior to transduction and by microinjection of lentivirus particles under the intact zona layer. Efficiency of transgenesis was high using both methods, up to 80% and 100% respectively. However, survival of the treated embryos was low in the case of zona removal techniques (10-20%), compared to 40-60% for microinjection. We are currently using transgenesis by lentiviral vectors expressing small interfering RNAs to generate knockdown mice to study functional genomics.