300 DEVELOPMENT OF TETRACYCLINE-REGULATED GREEN FLUORESCENT PROTEIN EXPRESSING TRANSGENIC RATS BY LENTIVIRAL VECTOR

Abstract

Although various methodologies have been available for genetic modification in mouse, such genomic tools are very limited in rats. The aim of the present study was to take advantage of lentiviral gene delivery to develop effective inducible germline transgenic rats. A tetracycline inducible system was designed to control the expression of the green fluorescent protein (GFP) gene. The GFP gene was constructed downstream of tetracycline response element under the control of a mini cytomegalovirus promoter. Additionally, rtTA2s-Ms was constructed at the 3 prime terminus of GFP under the control of an ubiquitin promoter. The resulted construct was named pLV-Tet-GFP. The pLV-Tet-GFP lentiviral vectors were injected into perivitelline space of 115 Sprague-Dawley rat zygotes. Of those 115 zygotes injected, 93 zygotes were transferred into 4 surrogate mothers and 32 live pups were obtained. The presence of transgene in Fo generation was first detected by PCR and subsequently Southern blot analysis in 25 out of 32 pups (78%). A varying level of GFP expression was observed in the 14 Fo pups after feeding them with 2 g L^–1 of tetracycline via drinking water for 4 days. Northern blot analysis was performed in the subsequent generations to determine spatial (brain, kidney, liver, and lung tissues) and temporal (day 0, day 4, day 11, and day 30) expression. The results showed expression intensity as being highest in kidney, brain, lung, and liver. There was little expression detected on day 11 and 30. This study suggests that lentiviral vectors can be used to deliver inducible system quite efficiently in rats.

The authors thank Dr. Carlos Lois for providing the lentiviral backbone, Dr. W. Hillen for providing the rtTA2s-Ms fragment, and GuoFu Fang for developing the inducible lentiviral vector.