title: Preimplantation screening for transgenesis using an embryonic specific promoter and green fluorescent protein.
abstract: We report enrichment in the efficiency of generating mice transgenic for expression of a human protein in their milk using GFP-mediated preimplantation screening. The transgene array consisted of a functional gene (human alpha-1 antitrypsin under the control of the ovine BLG promoter) linked 5' to a reporter gene (GFP under the control of the murine Oct-4 promoter). GFP expression was detected in blastocysts by fluorescence microscopy and green and nongreen embryos were transferred to recipients in separate groups. In the first experiment, of seven pups that resulted from the transfer of blastocysts expressing GFP, five (71%) were transgenic. The experiment was repeated and of 12 pups that resulted from transfer of GFP-expressing blastocysts, 11 were transgenic (92%). The presence of the reporter cassette used for preimplantation screening did not affect the expression level of alpha-1-antitrypsin in the milk of the transgenic mice. In addition, in a related experiment wherein the GFP reporter gene was co-injected with a second mammary-specific transgene, pINC, no effect on transgene expression was observed. For mice transgenic for the mammary-specific gene alone, expression levels for four different lines were 192, 197, 382, and 415 microg/mL. For mice transgenic for both the mammary-specific transgene and the Oct4-GFP reporter cassette, expression levels for seven different lines were 282, 321, 468, 497, 499, 516, and 806 microg/mL.
authors.affiliation: PPL Therapeutics, Inc., Blacksburg, Virginia, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Keiser
authors.forename: P M
authors.initials: PM
authors.lastname: Jobst
authors.forename: A S
authors.initials: AS
authors.lastname: Garst
authors.forename: J T
authors.initials: JT
authors.lastname: Boone
authors.forename: C B
authors.initials: CB
authors.lastname: Geyer
authors.forename: C
authors.initials: C
authors.lastname: Phelps
authors.forename: D L
authors.initials: DL
authors.lastname: Ayares
authors.forename: R L
authors.initials: RL
authors.lastname: Page
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins; D006801:Humans; D007782:Lactoglobulins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics* / Q000502:physiology; D050814:Octamer Transcription Factor-3; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D014157:Transcription Factors; D000515:alpha 1-Antitrypsin / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D007782:Lactoglobulins; D008164:Luminescent Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors; D000515:alpha 1-Antitrypsin; D049452:Green Fluorescent Proteins