Specific Knockdown of Gene Expression in the Mature Rat Pineal Gland: The Cone-Rod Homeodomain Transcription Factor...

Melatonin is synthesised from tryptophan by the sequential action of enzymes that are highly expressed in the pineal gland. Homeobox gene-encoded transcription factors typically control organ development; however, a set of homeobox genes is strongly expressed in the adult pineal gland. Previous in vitro experiments revealed that knockdown of homeobox genes in rat pinealocyte cultures reduced expression of melatonin-synthesising enzymes. Until now, it was not possible to determine the impact of homeobox genes on melatonin synthesis in vivo, which is needed to evaluate physiological functions. Using the cone-rod homeobox (Crx) gene as an example, we therefore developed an experimental pipeline to deliver short-hairpin RNA, via adeno-associated viral vectors, into the pineal gland of adult rats. This approach enabled us to selectively reduce Crx expression in the mature pineal gland, which we confirmed at both the transcript and protein levels. We employed a common approach in pharmacology to correlate Crx knockdown with the expression level of the tagged fluorescent reporter, which provided a quantitative basis to define data exclusion/inclusion criteria. Our efforts confirmed that knockdown of Crx in vivo reduced the expression of two melatonin-synthesising enzymes, namely tryptophan hydroxylase 1 and acetylserotonin O-methyltransferase, consistent with in vitro data. Furthermore, knockdown of pineal Crx significantly reduced nighttime plasma melatonin levels. Our work demonstrates a method through which knockdown of target genes in the rat pineal gland can be achieved without the need for transgenic models.