Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains

PLoS One. 2016 Aug 29;11(8):e0162023. doi: 10.1371/journal.pone.0162023. eCollection 2016.

Abstract

Adeno-associated virus (AAV) vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb) were obtained by progressively deleting the original 2.0-kb promoter from the 5' end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength) and 0.2-kb (70% astrocyte specificity) promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.

MeSH terms

  • Animals
  • Brain / metabolism*
  • Callithrix
  • Cerebellum / metabolism
  • Genetic Vectors / genetics
  • Glial Fibrillary Acidic Protein / genetics*
  • HEK293 Cells
  • Humans
  • Immunohistochemistry
  • Lentivirus / genetics
  • Mice
  • Mice, Inbred C57BL
  • Promoter Regions, Genetic / genetics*

Substances

  • Glial Fibrillary Acidic Protein

Grant support

This research is (partially) supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and the Japan Agency for Medical Research and Development, AMED, to HH and the MEXT KAKENHI (15K18330) to AK.