title: Dual promoter lentiviral vector generates transgenic mice expressing E2-CSFV glycoprotein in their milk, but impairs early identification of transgenic embryos.
abstract: Lentiviral vectors containing the green fluorescent protein gene have been successfully used to select transgenic embryos before transfer to a surrogate mother. However, there are apparently no reports regarding early detection of transgenic embryos using a lentiviral vector carrying an additional transcription unit for tissue-specific expression of a valuable protein. In this study, two HIV-based lentiviral vectors were constructed. The first one contained the green fluorescent protein (GFP) coding sequence driven by the early SV40 promoter (Lv-G), whereas the other contained an additional transcription unit for the expression of E2 glycoprotein from classical swine fever virus, driven by a 1.5 kb αS1casein promoter from water buffalo (Lv-αS1cE2hisG). Microinjection of single-cell mouse embryos with Lv-G lentiviral vector rendered embryos which were GFP-positive, beginning at the four-cell stage. Of 33 mice born, 28 (81%) carried the transgene DNA and 15 (55.5%) were GFP-positive. Microinjection of Lv-αS1cE2hisG lentiviral vector yielded 28 mice born; although 24 (85%) carried the transgene DNA, none were GFP-positive, suggesting that the tissue-specific expression cassette interfered with expression of the ubiquitous trancriptional unit. In Lv-αS1cE2hisG transgenic mice, E2his was expressed in milk as a homodimer (at concentrations ≤ 0.422 mg/mL). This was apparently the first report of expression of a recombinant protein in the milk of transgenic animals generated by lentiviral transgenesis.
authors.affiliation: Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Chile. osanchez@udec.cl
authors.forename: Oliberto Sánchez
authors.initials: OS
authors.lastname: Ramos
authors.forename: Yanet Prieto
authors.initials: YP
authors.lastname: Carratalá
authors.forename: Silvia Gómez
authors.initials: SG
authors.lastname: Puerta
authors.forename: Natalie C Parra
authors.initials: NC
authors.lastname: Pereira
authors.forename: Lester Suárez
authors.initials: LS
authors.lastname: Amarán
authors.forename: Silvana P Jimenez
authors.initials: SP
authors.lastname: Chaves
authors.forename: Jorge R Toledo
authors.initials: JR
authors.lastname: Alonso
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005260:Female; D018014:Gene Transfer Techniques / Q000009:adverse effects; D005822:Genetic Vectors / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000196:embryology*; D008892:Milk / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D014759:Viral Envelope Proteins; C087086:glycoprotein E2, classical swine fever virus