Transgenic Research

, Volume 16, Issue 5, pp 571–580

New technology for an old favorite: lentiviral transgenesis and RNAi in rats



The ability to produce targeted deletions in the mouse genome via homologous recombination has been a hallmark of mouse genetics, and has lead to the production of thousands of gene knockouts. New technologies are making it possible to disrupt gene function in many other species. This article reviews some of these methods, highlighting the powerful combination of lentiviral vectors with RNA interference (RNAi), which allows one to produce transgenic animals expressing short hairpin RNA (shRNA) to “knock down” specific gene expression. Lentiviral transduction of embryos has been shown to be a highly efficient means of transgenesis, and is particularly promising for animals that are considered difficult to genetically modify by DNA pronuclear injection. This technique has been popular for introducing transgenes for shRNA expression into rodents and its utility for creating new genetic models has already been demonstrated. One of the purported advantages of in vivo RNAi is that shRNA expressing transgenes would be expected to act in a dominant nature, resulting in a phenotype in founder animals. However, one possible concern with lentiviral-mediated transgenesis is the potential for mosaicism in founders, and the data for this phenomenon and the potential causes and solutions are discussed. Emphasis is placed on the application of in vivo RNAi, and other reverse genetic methods, for creating new genetic models in the rat.


Lentiviral transgenesis Gene knockdown shRNA RNAi Reverse genetics Rat 


  1. Babcock AM, Standing D, Bullshields K, Schwartz E, Paden CM, Poulsen DJ (2005) In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference. Mol Ther 11:899–905PubMedCrossRefGoogle Scholar
  2. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R (2003) Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 34:263–264PubMedCrossRefGoogle Scholar
  3. Cao W, Hunter R, Strnatka D, McQueen CA, Erickson RP (2005) DNA constructs designed to produce short hairpin, interfering RNAs in transgenic mice sometimes show early lethality and an interferon response. J Appl Genet 46:217–225PubMedGoogle Scholar
  4. Capecchi MR (1989) Altering the genome by homologous recombination. Science 244:1288–1292PubMedCrossRefGoogle Scholar
  5. Carmell MA, Zhang L, Conklin DS, Hannon GJ, Rosenquist TA (2003) Germline transmission of RNAi in mice. Nat Struct Biol 10:91–92PubMedCrossRefGoogle Scholar
  6. Chen W, Liu M, Jiao Y, Yan W, Wei X, Chen J, Fei L, Liu Y, Zuo X, Yang F, Lu Y, Zheng Z (2006) Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo. J Virol 80:3559–3566PubMedCrossRefGoogle Scholar
  7. Collidge TA, Lammie GA, Fleming S, Mullins JJ (2004) The role of the renin-angiotensin system in malignant vascular injury affecting the systemic and cerebral circulations. Prog Biophys Mol Biol 84:301–319PubMedCrossRefGoogle Scholar
  8. Coumoul X, Shukla V, Li C, Wang RH, Deng CX (2005) Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference. Nucleic Acids Res 33:e102PubMedCrossRefGoogle Scholar
  9. Cullen BR (2006) Enhancing and confirming the specificity of RNAi experiments. Nat Methods 3:677–681PubMedCrossRefGoogle Scholar
  10. Dai F, Yusuf F, Farjah GH, Brand-Saberi B (2005) RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis. Dev Biol 285:80–90PubMedCrossRefGoogle Scholar
  11. Dann CT, Alvarado AL, Hammer RE, Garbers DL (2006) Heritable and stable gene knockdown in rats. Proc Natl Acad Sci USA 103:11246–11251PubMedCrossRefGoogle Scholar
  12. Fan L, Moon J, Crodian J, Collodi P (2006) Homologous recombination in zebrafish ES cells. Transgenic Res 15:21–30PubMedCrossRefGoogle Scholar
  13. Fedoriw AM, Stein P, Svoboda P, Schultz RM, Bartolomei MS (2004) Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303:238–240PubMedCrossRefGoogle Scholar
  14. Golding MC, Long CR, Carmell MA, Hannon GJ, Westhusin ME (2006) Suppression of prion protein in livestock by RNA interference. Proc Natl Acad Sci USA 103:5285–5290PubMedCrossRefGoogle Scholar
  15. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541PubMedCrossRefGoogle Scholar
  16. Hammer RE, Maika SD, Richardson JA, Tang JP, Taurog JD (1990) Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human beta 2m: an animal model of HLA-B27-associated human disorders. Cell 63:1099–1112PubMedCrossRefGoogle Scholar
  17. Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE, Garbers DL (2005) Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc Natl Acad Sci USA 102:17430–17435PubMedCrossRefGoogle Scholar
  18. Hamra FK, Gatlin J, Chapman KM, Grellhesl DM, Garcia JV, Hammer RE, Garbers DL (2002) Production of transgenic rats by lentiviral transduction of male germ-line stem cells. Proc Natl Acad Sci USA 99:14931–14936PubMedCrossRefGoogle Scholar
  19. Hasuwa H, Kaseda K, Einarsdottir T, Okabe M (2002) Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 532:227–230PubMedCrossRefGoogle Scholar
  20. Hedrich H (2000) History, strains and models. Academic Press, LondonGoogle Scholar
  21. Hofmann A, Zakhartchenko V, Weppert M, Sebald H, Wenigerkind H, Brem G, Wolf E, Pfeifer A (2004) Generation of transgenic cattle by lentiviral gene transfer into oocytes. Biol Reprod 71:405–409PubMedCrossRefGoogle Scholar
  22. Homberg JR, Olivier JD, Smits BM, Mul JD, Mudde J, Verheul M, Nieuwenhuizen OF, Cools AR, Ronken E, Cremers T, Schoffelmeer AN, Ellenbroek BA, Cuppen E (2007) Characterization of the serotonin transporter knockout rat: A selective change in the functioning of the serotonergic system. Neuroscience 146(4):1662–1676PubMedCrossRefGoogle Scholar
  23. Hou J, Shan Q, Wang T, Gomes AS, Yan Q, Paul DL, Bleich M, Goodenough DA (2007) Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium. J Biol Chem 282(23):17114–17122PubMedCrossRefGoogle Scholar
  24. Ikawa M, Tanaka N, Kao WW, Verma IM (2003) Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther 8:666–673PubMedCrossRefGoogle Scholar
  25. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637PubMedCrossRefGoogle Scholar
  26. Kanatsu-Shinohara M, Ikawa M, Takehashi M, Ogonuki N, Miki H, Inoue K, Kazuki Y, Lee J, Toyokuni S, Oshimura M, Ogura A, Shinohara T (2006) Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc Natl Acad Sci USA 103:8018–8023PubMedCrossRefGoogle Scholar
  27. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69:612–616PubMedCrossRefGoogle Scholar
  28. Kanatsu-Shinohara M, Toyokuni S, Shinohara T (2005) Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells. Biol Reprod 72:236–240PubMedCrossRefGoogle Scholar
  29. Kittler R, Putz G, Pelletier L, Poser I, Heninger AK, Drechsel D, Fischer S, Konstantinova I, Habermann B, Grabner H, Yaspo ML, Himmelbauer H, Korn B, Neugebauer K, Pisabarro MT, Buchholz F (2004) An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432:1036–1040PubMedCrossRefGoogle Scholar
  30. Kubota H, Avarbock MR, Brinster RL (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA 101:16489–16494PubMedCrossRefGoogle Scholar
  31. Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J (2003) Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol 21:559–561PubMedCrossRefGoogle Scholar
  32. Lillico SG, Sherman A, McGrew MJ, Robertson CD, Smith J, Haslam C, Barnard P, Radcliffe PA, Mitrophanous KA, Elliot EA, Sang HM (2007) Oviduct-specific expression of two therapeutic proteins in transgenic hens. Proc Natl Acad Sci USA 104:1771–1776PubMedCrossRefGoogle Scholar
  33. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868–872PubMedCrossRefGoogle Scholar
  34. Lu W, Yamamoto V, Ortega B, Baltimore D (2004) Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell 119:97–108PubMedCrossRefGoogle Scholar
  35. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348–352PubMedCrossRefGoogle Scholar
  36. Miskevich F, Doench JG, Townsend MT, Sharp PA, Constantine-Paton M (2006) RNA interference of Xenopus NMDAR NR1 in vitro and in vivo. J Neurosci Methods 152:65–73PubMedCrossRefGoogle Scholar
  37. Mullins JJ, Peters J, Ganten D (1990) Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature 344:541–544PubMedCrossRefGoogle Scholar
  38. Nakagawa T, Feliu-Mojer MI, Wulf P, Lois C, Sheng M, Hoogenraad CC (2006) Generation of lentiviral transgenic rats expressing glutamate receptor interacting protein 1 (GRIP1) in brain, spinal cord and testis. J Neurosci Methods 152:1–9PubMedCrossRefGoogle Scholar
  39. Persengiev SP, Zhu X, Green MR (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10:12–18PubMedCrossRefGoogle Scholar
  40. Pfeifer A (2004) Lentiviral transgenesis. Transgenic Res 13:513–522PubMedCrossRefGoogle Scholar
  41. Pfeifer A, Ikawa M, Dayn Y, Verma IM (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 99:2140–2145PubMedCrossRefGoogle Scholar
  42. Rao MK, Pham J, Imam JS, MacLean JA, Murali D, Furuta Y, Sinha-Hikim AP, Wilkinson MF (2006) Tissue-specific RNAi reveals that WT1 expression in nurse cells controls germ cell survival and spermatogenesis. Genes Dev 20:147–152PubMedCrossRefGoogle Scholar
  43. Reynolds A, Anderson EM, Vermeulen A, Fedorov Y, Robinson K, Leake D, Karpilow J, Marshall WS, Khvorova A (2006) Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12:988–993PubMedCrossRefGoogle Scholar
  44. Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Ihrig MM, McManus MT, Gertler FB, Scott ML, Van Parijs L (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33:401–406PubMedCrossRefGoogle Scholar
  45. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P, Dorin J, Cooke HJ (1997) The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389:73–77PubMedCrossRefGoogle Scholar
  46. Ryu BY, Kubota H, Avarbock MR, Brinster RL (2005) Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Natl Acad Sci USA 102:14302–14307PubMedCrossRefGoogle Scholar
  47. Seibler J, Kleinridders A, Kuter-Luks B, Niehaves S, Bruning JC, Schwenk F (2007) Reversible gene knockdown in mice using a tight, inducible shRNA expression system. Nucleic Acids Res 35:e54PubMedCrossRefGoogle Scholar
  48. Seibler J, Kuter-Luks B, Kern H, Streu S, Plum L, Mauer J, Kuhn R, Bruning JC, Schwenk F (2005) Single copy shRNA configuration for ubiquitous gene knockdown in mice. Nucleic Acids Res 33:e67PubMedCrossRefGoogle Scholar
  49. Shinagawa T, Ishii S (2003) Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes Dev 17:1340–1345PubMedCrossRefGoogle Scholar
  50. Smits BM, Mudde JB, van de Belt J, Verheul M, Olivier J, Homberg J, Guryev V, Cools AR, Ellenbroek BA, Plasterk RH, Cuppen E (2006) Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis. Pharmacogenet Genomics 16:159–169PubMedGoogle Scholar
  51. Tiscornia G, Singer O, Ikawa M, Verma IM (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 100:1844–1848PubMedCrossRefGoogle Scholar
  52. van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ (2006a) Germline transmission of genetically modified primordial germ cells. Nature 441:766–769PubMedCrossRefGoogle Scholar
  53. van de Lavoir MC, Mather-Love C, Leighton P, Diamond JH, Heyer BS, Roberts R, Zhu L, Winters-Digiacinto P, Kerchner A, Gessaro T, Swanberg S, Delany ME, Etches RJ (2006b) High-grade transgenic somatic chimeras from chicken embryonic stem cells. Mech Dev 123:31–41PubMedCrossRefGoogle Scholar
  54. van den Brandt J, Wang D, Kwon SH, Heinkelein M, Reichardt HM (2004) Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo. Genesis 39:94–99PubMedCrossRefGoogle Scholar
  55. von Horsten S, Schmitt I, Nguyen HP, Holzmann C, Schmidt T, Walther T, Bader M, Pabst R, Kobbe P, Krotova J, Stiller D, Kask A, Vaarmann A, Rathke-Hartlieb S, Schulz JB, Grasshoff U, Bauer I, Vieira-Saecker AM, Paul M, Jones L, Lindenberg KS, Landwehrmeyer B, Bauer A, Li XJ, Riess O (2003) Transgenic rat model of Huntington’s disease. Hum Mol Genet 12:617–624CrossRefGoogle Scholar
  56. Whitehurst AW, Bodemann BO, Cardenas J, Ferguson D, Girard L, Peyton M, Minna JD, Michnoff C, Hao W, Roth MG, Xie XJ, White MA (2007) Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 446:815–819PubMedCrossRefGoogle Scholar
  57. Xia X, Zhou H, Huang Y, Xu Z (2006a) Allele-specific RNAi selectively silences mutant SOD1 and achieves significant therapeutic benefit in vivo. Neurobiol Dis 23:578–586PubMedCrossRefGoogle Scholar
  58. Xia XG, Zhou H, Samper E, Melov S, Xu Z (2006b) Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet 2:e10PubMedCrossRefGoogle Scholar
  59. Yu J, McMahon AP (2006) Reproducible and inducible knockdown of gene expression in mice. Genesis 44:252–261PubMedCrossRefGoogle Scholar
  60. Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Hu R, Lopez-Guajardo CC, Brose HL, Porter KI, Leonard RA, Hitt AA, Schommer SL, Elegbede AF, Gould MN (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol 21:645–651PubMedCrossRefGoogle Scholar
  61. Zhou H, Falkenburger BH, Schulz JB, Tieu K, Xu Z, Xia XG (2007) Silencing of the Pink1 gene expression by conditional RNAi does not induce dopaminergic neuron death in mice. Int J Biol Sci 3:242–250PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Department of PharmacologyUniversity of Texas Southwestern Medical CenterDallasUSA
  2. 2.Cecil H. & Ida Green Center for Reproductive Biology SciencesUniversity of Texas Southwestern Medical CenterDallasUSA

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