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DOI: 10.1126/science.1067081 , 868 (2002); 295Science et al.Carlos Lois, Vectors Expression of Transgenes Delivered by Lentiviral Germline Transmission and Tissue-Specific www.sciencemag.org (this information is current as of August 4, 2008 ): The following resources related to this article are available online at http://www.sciencemag.org/cgi/content/full/295/5556/868 version of this article at: including high-resolution figures, can be found in the onlineUpdated information and services, http://www.sciencemag.org/cgi/content/full/1067081/DC1 can be found at: Supporting Online Material found at: can berelated to this articleA list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/295/5556/868#related-content 379 article(s) on the ISI Web of Science. cited byThis article has been http://www.sciencemag.org/cgi/content/full/295/5556/868#otherarticles 96 articles hosted by HighWire Press; see: cited byThis article has been http://www.sciencemag.org/cgi/collection/medicine Medicine, Diseases : subject collectionsThis article appears in the following http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprintsInformation about obtaining registered trademark of AAAS. is aScience2002 by the American Association for the Advancement of Science; all rights reserved. The title CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience o n A ug us t 4 , 2 00 8 w w w .s ci en ce m ag .o rg D ow nl oa de d fro m Hsp40. These data suggest a role for chap- erones in pathologies involving a- synuclein in humans, such that Hsp70 may be a critical part of the neuronal arsenal that mitigates a-synuclein toxicity. An alterna- tive interpretation is that the presence of chaperones in aggregates results in their cellular depletion, due to sequestration, and this loss of chaperone function leads to degeneration. We present data that implicates the molec- ular chaperone machinery in the pathogenesis of PD using a Drosophila model. Augmenta- tion of Hsp70 activity in vivo suppresses a-synuclein neurotoxicity, whereas compro- mising chaperone function enhances a-syn- uclein–induced dopaminergic neuronal loss. Thus, chaperone machinery in flies helps to protect dopaminergic neurons against degener- ation and attenuates the neurotoxic consequenc- es of a-synuclein expression. Hsp70 may mit- igate a-synuclein toxicity by influencing the conformation of a-synuclein in ways that are not revealed by the morphology of aggregates in Drosophila. Alternatively, a-synuclein may be toxic because it interferes with chaperone activity, possibly by their sequestration, and it is this effect that is mitigated by added Hsp70. Our findings suggest a role for chaperones in human pathology, because human LBs and LNs in PD and other human synucleinopathies immunostain for Hsp70 and Hsp40. Chaper- ones may thus play a role in a-synuclein tox- icity, such that augmentation of chaperone stress pathways may be an effective approach in the treatment of several human neurodegen- erative diseases including PD. References and Notes 1. A. E. Lang, A. M. Lozano, N. Engl. J. Med. 339, 1044 (1998). 2. M. G. Spillantini, R. A. Crowther, R. Jakes, M. Hase- gawa, M. Goedert, Proc. Natl. Acad. Sci. U.S.A. 95, 6469 (1998). 3. M. G. Spillantini et al., Nature 388, 839 (1997). 4. M. Baba et al., Am. J. Pathol. 152, 879 (1998). 5. M. H. Polymeropoulos et al., Science 276, 2045 (1997). 6. R. Kruger et al., Nature Genet. 18, 106 (1998). 7. M. B. Feany, W. W. Bender, Nature 404, 394 (2000). 8. G. R. Jackson et al., Neuron 21, 633 (1998). 9. J. M. Warrick et al., Cell 93, 939 (1998). 10. P. Fernandez-Funez et al., Nature 408, 101 (2000). 11. J. M. Warrick et al., Nature Genet. 23, 425 (1999). 12. B. Bukau, A. L. Horwich, Cell 92, 351 (1998). 13. J. R. Glover, S. Lindquist, Cell 94, 73 (1998). 14. Y. O. Chernoff, S. L. Lindquist, B. Ono, S. G. Inge- Vechtomov, S. W. Liebman, Science 268, 880 (1995). 15. A. H. Brand, N. Perrimon, Development 118, 401 (1993). 16. H. Li, S. Chaney, I. J. Roberts, M. Forte, J. Hirsh, Curr. Biol. 10, 211 (2000). 17. Drosophila were grown under standard conditions at 25¡C. Transgenes used included Ddc-GAL4 (16); UAS- lacZ; UAS-HspA1L encoding human Hsp70 (11). UAS- a-syn, UAS-A30P, and UAS-A53T were generated for a-synuclein; additional lines are described in (7). Male and female ßies were aged to the indicated time, then heads were Þxed in 10% neutral-buffered formalin (NBF) and embedded in parafÞn. Serial sec- tions (8 mm thickness) through the entire brain were prepared for immunostaining. Antibodies used were: TH (1:150, Pelfreez, Rogers, AR), human Hsp70 (SC- 24 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), Drosophila Hsp70 [7FB (21), 1:500], ubiquitin (MAB 1510, 1:2000, Chemicon, Temecula, CA), and a-synuclein [syn303 (26), 1:100 and 1:1000, at the latter dilution, syn303 selectively detects aggregated a-synuclein; syn514 (26), 1:5, which only detects aggregated a-synuclein]. Sections were incubated overnight with primary antibody at 4¡C, followed by secondary antibody, Avidin-Biotin Complex incuba- tion (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA), and developing with 3,39-diamino- benzidine. For each data point, complete serial sec- tions from three to Þve individual brains were exam- ined. Similar results were seen in at least Þve inde- pendent experiments. No sex differences were noted. The extent of neuron loss was less than previously reported (7), even using the same transgenic lines. We did not see effects of a-synuclein on climbing behavior. 18. Supplementary data are available at Science Online at www.sciencemag.org/cgi/content/full/1067389/ DC1. 19. Dopaminergic neuronal loss at 30 days was the same as at 20 days. 20. In the DL-1 clusters, zero to one inclusions were present at 1 day and two to three inclusions at 20 days. In the DM clusters, zero to one inclusions were observed at both 1 and 20 days. The number of inclusions was unaltered by Hsp70. 21. J. M. Velazquez, S. Lindquist, Cell 36, 655 (1984). 22. F. Elefant, K. B. Palter, Mol. Biol. Cell 10, 2101 (1999). 23. Y. Imai et al., Cell 105, 891 (2001). 24. H. Shimura et al., Science 293, 263 (2001). 25. Blocks of cingulate cortex, amygdala, and midbrain from postmortem PD, LBVAD, DLB, and NBIA1 brains were Þxed in 70% ethanol/150 mM NaCl or 10% NBF and embedded in parafÞn. sections (6 mm thick- ness) were cut and stained (17). Immunostaining was with a-synuclein antibodies as in (17), and human Hsp70 (SC-24, Santa Cruz Biotechnologies; SPA-810, StressGen Biotechnologies), Hsp40 (SC-1801, Santa Cruz Biotechnologies), and HDJ-2 (KA2A5.6, NeoMar- kers, Fremont, CA). 26. B. I. Giasson et al., J. Neurosci. Res. 59, 528 (2000). 27. We thank M. Feany, J. Hirsh, S. Lindquist, and K. Palter for sharing reagents, B. Giasson and T. Schuck for their expertise, and A. Cashmore and L. Lillien for critical reading of the manuscript. We thank the reviewers for insightful comments. This research was funded, in part, by a Pioneer award from the Alzhei- merÕs Association ( J.Q.T. and V.M.-Y.L.), the Well- come Trust (H.Y.E.C.), a Developmental Biology Training Grant and National Research Service Award (P.K.A.), the David and Lucile Packard Foundation (N.M.B.), and the National Institute on Aging. N.M.B is an assistant investigator of the Howard Hughes Medical Institute. 24 October 2001; accepted 11 December 2001 Published online 20 December 2001; 10.1126/science.1067389 Include this information when citing this paper. Germline Transmission and Tissue-Specific Expression of Transgenes Delivered by Lentiviral Vectors Carlos Lois,* Elizabeth J. Hong,* Shirley Pease, Eric J. Brown, David Baltimore† Single-cell mouse embryos were infected in vitro with recombinant lentiviral vectors to generate transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter. Eighty percent of founder mice carried at least one copy of the transgene, and 90% of these expressed GFP at high levels. Progeny inherited the transgene(s) and displayed green fluorescence. Mice generated using lentiviral vectors with muscle-specific and T lymphocyte–specific promoters expressed high levels of GFP only in the appropriate cell types. We have also generated transgenic rats that express GFP at high levels, suggesting that this technique can be used to produce other transgenic animal species. The ability to introduce and express exoge- nous genes of interest in animals has become an indispensable tool to modern biologists (1). Transgenic mice are currently generated by pronuclear injection; however, this tech- nique is still relatively inefficient, technically demanding, costly, and impractical in most other animal species. Another approach to transgenesis is to use retroviruses as gene delivery vehicles because they are able to stably integrate into the genome of cells. However, the generation of transgenic ani- mals with oncoretroviruses such as the Molo- ney murine leukemia virus (MoMLV) is im- practical because silencing of the provirus during development results in low to unde- tectable levels of transgene expression (2, 3). Lentiviruses are a class of retroviruses that cause chronic illnesses in the host organ- isms they infect. Among retroviruses, lenti- viruses have the distinguishing property of being able to infect both dividing and nondi- viding cells, and this ability has led to their development as gene delivery vehicles (4 ). Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. *These authors contributed equally to this work. †To whom correspondence should be addressed. E- mail: baltimo@caltech.edu R E P O R T S 1 FEBRUARY 2002 VOL 295 SCIENCE www.sciencemag.org868 o n A ug us t 4 , 2 00 8 w w w .s ci en ce m ag .o rg D ow nl oa de d fro m To determine whether, in contrast to oncoret- roviruses, lentiviruses might be immune to developmental silencing, we used lentiviral- based vectors to generate transgenic mice and rats. The lentiviral backbone used in these experiments is based on a self-inactivating vector described previously (5, 6 ) (Fig. 1, top). The vector was engineered to carry an internal promoter driving the GFP reporter gene. After testing several promoters, the human ubiquitin-C promoter was found to provide the most reliable expression across different cell types and was selected for subsequent experiments (7–11). To in- crease the level of transcription, the wood- chuck hepatitis virus posttranscriptional regulatory element (WRE) was inserted downstream of GFP (12). To increase the titer of the virus, the human immunodefi- ciency virus–1 (HIV-1) flap element (13) was inserted between the 59 long terminal repeat (LTR) and the human ubiquitin-C internal promoter to generate the viral vec- tor called FUGW (see Fig. 1, top). Viruses were pseudotyped with the vesicular stoma- titis virus glycoprotein (VSVG) and con- centrated by ultracentrifugation to approx- imately 1 3 106 infectious units (I.U.)/ml. Approximately 10 to 100 pl of concen- trated virus was injected into the periv- itelline space of single-cell mouse embryos (14–16 ). After 72 hours in culture, GFP expression was apparent in the blastula- or morula-stage embryos developing from the infected zygotes [see supplementary data (17 )]. Embryos were implanted into pseu- dopregnant females and were carried to term (15). In an initial trial, Southern blot analysis showed that 14 (82%) of 17 founder animals carried at least one copy of the integrated transgene (18). GFP fluores- cence, indicating expression from the trans- gene, was seen in the paws, tails, and face of 13 (76%) of these founder animals (18) (Fig. 1, A and B). In a second trial, 49 (87.5%) of 56 founder animals carried at least one copy of the transgene, and 45 (80%) of the founders expressed GFP (Ta- ble 1). All GFP-positive animals carried an integrated provirus, and all animals with two or more copies of the provirus ex- pressed the transgene at levels detectable by direct viewing of GFP fluorescence. The intensity of GFP fluorescence correlated positively with copy number, as estimated qualitatively (17 ). All major tissues and organs, including skin, bone, skeletal mus- cle, cardiac muscle, lung, liver, thymus, spleen, stomach, intestine, kidney, brain, retina, and gonads, were GFP-positive (see Fig. 1, C through F, for a representative data set). The delivery of the virus by injection into the perivitelline space yielded transgenics with high efficiency; however, the number of integrated proviruses in the genome varied substantially from animal to animal, ranging from 0 to more than 20 (17 ). A likely source of this variability is difficulty in controlling the volume of virus delivered into the peri- vitelline space during the injection. As an Fig. 1. GFP is expressed in embryos and the tissues of adult mice derived from the infection of zygotes with the FUGW lentiviral vector. (Top) Diagram of the lentiviral vector FUGW used to generate transgenic mice and rats. Only the relevant portions of the plasmid are shown. All vectors have the CMV enhancer substituted for the U3 region of the 59 LTR (pCL conÞguration) to maximize expression of viral RNA genomes during transient transfection (29). DU3 denotes a deletion in the U3 region of the 39 LTR that renders the 59 LTR of the integrated provirus transcriptionally inactive (5). The positions of the restriction sites Pst I and Bam HI used for Southern blot analysis of proviral copy number are indicated. (Bottom) Expression of GFP in the (A) face, (B) paw, (C) brain, (D) heart, (E) liver, and (F) kidney of a transgenic founder. The animal shown here carried eight proviral insertions. The animal was killed at 6 weeks of age by an overdose of anesthesia, intracardially perfused with Þxative, and viewed immediately under a ßuorescent dissecting microscope. A wild-type animal, identically prepared and photographed, is included for comparison. BF, brightÞeld photograph; WT and TG, ßuorescent images of wild-type control and transgenic animals, respectively. Table 1. Embryo viability and rates of implantation, transgenesis, and expression. M, mouse; FUGW, ubiquitin C promoter-GFP; FMHGW, myogenin promoterÐ H2B-GFP; PV, perivitelline injection; CI, viral co-incubation. The range and average number of proviral insertions for each experimental group is determined only from those animals that are transgenic, that is, the animals that carry one or more copies of the transgene. Animal Viral construct Method Virus conc. 3103 (I.U./ml) No. embryos No. animals No. copies Treated Viable Implanted Born Transgenic Expressing Average Range M FUGW PV (trial 1) 103 117 81 78 17 14 13 6.2 1Ð12 M FUGW PV (trial 2) 103 153 150 119 56 49 45 9 1Ð21 M FUGW CI 20 45 Ð* 29 5 5 5 7.2 2Ð12 M FUGW CI 4 (trial 1) 25 Ð* 18 7 5 5 3.8 2Ð7 M FUGW CI 4 (trial 2) 120 Ð* 59 11 8 7 2.6 1Ð5 M FUGW CI 0.8 75 Ð* 40 8 1 1 1 Ñ M FMHGW PV 103 106 86 74 15† 11 3 of 7‡ 4.8 2Ð15 Rat FUGW PV 103 233 210 130 22 13 9 3.3 1Ð7 *See (16). †Six founder embryos were recovered at 11.5 dpc (see text). ‡GFP expression can be determined only after sacriÞcing these animals. Data is unavailable from animals kept for breeding. R E P O R T S www.sciencemag.org SCIENCE VOL 295 1 FEBRUARY 2002 869 o n A ug us t 4 , 2 00 8 w w w .s ci en ce m ag .o rg D ow nl oa de d fro m alternative procedure, we removed the zona pellucidae and incubated the denuded embry- os with the lentiviral suspension at 2 3 104 I.U./ml, 4000 I.U./ml, and 800 I.U./ml (16 ). Embryos were cultured in vitro for approxi- mately 3 days to the morula or blastocyst stage, when they were implanted into the uterus of timed pseudopregnant females (15). Denuded embryos were delayed in their de- velopment in vitro with respect to their un- treated counterparts; furthermore, the rate of implantation was lower than that of virus- injected embryos implanted with intact zona pellucidae (18 versus 38%). All animals de- veloping from embryos incubated with 2 3 104 I.U./ml carried at least six proviral inte- grations, two (28.6%) of seven animals de- veloping from embryos incubated with 4000 I.U./ml carried one or two copies of the pro- virus, and one of eight animals derived from embryos incubated with 800 I.U./ml carried the transgene. A second trial with 4000 I.U./ ml gave comparable results (Table 1). Al- though there is still some nonlinearity and irreproducibility, this method of virus deliv- ery allows for better control of the number of proviral integrations per genome. Further- more, incubating embryos in a virus-contain- ing solution is a process that requires no specialized equipment and may be easier for many laboratories that wish to use this technique. Founders carrying transgene(s) transmit- ted most of them to a fraction of their prog- eny (18) (Fig. 2). The bands corresponding to the proviral insertions characteristic of the founder animals segregated among the prog- eny. In the Southern blots of the founder animals, we occasionally observed animals with bands of varying intensity, suggesting they were genetic mosaics (see arrow in Fig. 2). In contrast, the intensity of the bands corresponding to the insertions in the progeny was uniform. Furthermore, ubiquitous GFP expression similar to that of the founder an- imals was observed in transgenic F1 progeny, indicating that the provirus was not inactivat- ed through one round of gametogenesis and development (17 ). All animals carrying two or more insertions of the FUGW provirus expressed GFP at levels detectable by direct fluorescence. However, among transgenic lines carrying one proviral insert, approxi- mately half expressed the transgene at levels detectable by direct fluorescence (Fig. 2). In one single-insertion line in which GFP ex- pression was not observed by direct viewing, GFP was detectable by Western blot analysis in some tissues (brain, testes), but not in others (heart, lung, liver, kidney, spleen, skel- etal muscle) (17, 18). This suggests that the specific genomic locus into which an individ- ual provirus has integrated may affect the transcriptional activity of some transgenes delivered by this method. To determine whether lentiviral vectors could be used to express genes in a tissue- specific manner, we engineered a viral vector, FMHGW, in which a histone 2B–GFP (H2B- GFP) fusion gene was driven by the myogenin promoter, the activity of which is specific to skeletal muscle (19). The H2B-GFP reporter was used to concentrate the fluorescence in the nuclei, making the signal more intense (20, 21). Transgenic animals were generated with the FMHGW viral vector by delivering the lentivi- rus into the perivitelline space of single-cell embryos, as described above (16). Two of the six embryos recovered at day 11.5 of pregnancy showed GFP fluorescence in the paraxial and cephalic somites, limb buds, and extraocular muscles in the pattern expected for the myoge- nin protein muscle (19) (Fig. 3). Immunofluo- rescence of frozen tissue sections with an anti- body raised against GFP showed that expres- sion was limited to the nuclei of cells in the skeletal muscle lineage, whereas cells of the skin, cartilage, neural tube, heart, lung, and intestines were negative (18) (Fig. 3, C and D). Southern blot analysis of these embryos showed that although all six embryos were transgenic, only those animals carrying six or more copies of the proviral insert expressed at levels detectable by direct GFP fluorescenc