Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue-specific gene expression.
Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.
Sato
M
M
Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan. massato@is.icc.u-tokai.ac.jp
Watanabe
T
T
Oshida
A
A
Nagashima
A
A
Miyazaki
J I
JI
Kimura
M
M
D000199:Actins / Q000235:genetics; D000818:Animals; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D004676:Myelin Basic Protein / Q000235:genetics; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
D000199:Actins; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D004676:Myelin Basic Protein; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins