Functional Investigation of Human Limb Specific Enhancer in Transgenk Zebrafish Assay

Abstract

The diverse morphogenic programs exploited during embryonic development depends on the precise expression of a large number of genes in time and space. Genetic studi es suggested that stereotypic development of embryos is driven by large interconnected regulatory networks within non-coding part of genome that define the precise patterns of gene expression. Most of the human genome is transcribed, but only a small percentage has coding information imbedded. The rest of the 97% genome, the non-coding genome, labeled as 'junk DNA', is where evolutionary complexity resides. Enhancers, short stretches of non-coding DNA, confer spatial and temporal specificity to gene expression patterns. Dispersal of enhancers across vast regions of vertebrate genome makes it difficult to define enhancer-gene association, which is significant to effectively analyze the importance of enhancers in development, disease, and evolution. In line with these observations, O,1hology mapping approach was previously done to compare enhancer expression to the endogenous expression pattern of neighboring genes (Tbx4 and NACA2). It also showed that the enhancer can be a key regulator of limb-specific adjacent genes. Additionally, cis-regulatory element chr17 crm 47 (GRCh38:17:61574768:61575752:1) possessed a human-specific single nucleotide variation that was linked with hip osteoarthritis. This study is an attempt to functionally validate the human limb specific enhancer. For the purpose, we opted an approach for the formation of an entry clone vector. This study focuses on functional investigation of selected chr17_crm_47 in vivo. We devised a methodology in which chr17 _crm_ 47 flanking regions (amplicon size 1500bp) were amplified from human genomic DNA through PCR then the PCR product was purified and quantified. Furthermore, bacterial culture was prepared having Spectinomycin antibacterial resistance; E.coli DH5a competent cells were prepared and pCR8/GW/TOPO/TA cloning kit was used to clone the element, chr17 _crm_ 47. The purified plasmid was confirmed through EcoRl digestion and Orientation PCR. Thus, entry vector having attL recombination sites is generated by using Topo cloning. To conclude, elucidation of cis-acting regulatory elements can be used as a powerful tool to gain insight of the regulatory role of the chrl7 _crm_ 47 in early embryogenesis. Additionally, these cis-regulatory circuits are excellent candidates for TBX4 associated disorders caused by mutations, which will open up new opportunities for both disease detection and genetic therapy.