title: Generation of Knock down Tools for Transcription Factor 7-like-2 (TCF7L2) and Evaluation of its Expression Pattern in Developing Chicken Optic Tectum.
abstract: Backgroud: TCF7L2 encodes for a transcription factor that plays a significant role in the Wnt signaling pathway and has been implicated in schizophrenia and blood glucose homeostasis. During embryonic development, TCF7L2 is expressed widely in the midbrain and forebrain regions of mouse brain. However, expression data of TCF7L2 and its functional studies are much scarcer for chicken brain. Our study demonstrated interesting expression pattern of TCF7L2 in the optic tectum of developing chicken embryo. Further study was undertaken to develop and test the knock down tools for TCF7L2 in the developing chicken embryo which would help in its functional characterization.
  
  
  
  An RNAi based strategy was chosen to knock down TCF7L2A. miRNA based construct was designed and developed to target TCF7L2. However these miRNA construct may or may not be effective in knocking down their target gene, therefore the efficiency of the miRNA construct in knocking down TCF7L2 was tested. First the efficiency was measured in vitro using a sensor assay. The efficient pre-miRNA was cloned in an avian retrovirus to achieve in vivo knock down in the developing chicken embryo. RCAS-miRNA construct targeting TCF7L2, was electroporated in the tectum of embryonic day 3.5 chicken embryo. The extent of in vivo knock down of TCF7L2 by the RCAS-miRNA-TCF7L2 was examined using RNA in situ hybridization technique.
  
  
  
  TCF7L2 showed strong layer specific expression from embryonic day 5 to embryonic day 13 which is also the important time window for tectal development. In addition to the layer specific expression, TCF7L2 is also strongly expressed in the pre-tegmentum area of the tectum. The TCF7L2 transcript showed marked decrease in the region that was electroporated with RCAS-miRNA-TCF7L2.
  
  
  
  Therefore this study has led to the generation and validation of RCAS-pre miRNA-TCF7L2 which effectively knocks down TCF7L2 in vivo and thus has paved the way for further functional characterization of TCF7L2 in the chicken optic tectum.
authors.forename: Nashrah
authors.initials: N
authors.lastname: Ahmad
authors.affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, U.P., India. nashrah@iitk.ac.in.
authors.forename: Shabana
authors.initials: S
authors.lastname: Bano