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Construction and Quantitative Validation of Chicken CXCR4 Expression Reporter

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Abstract

Site directional migration is an important biological event and an essential behavior for latent migratory cells. A migratory cell maintains its motility, survival, and proliferation abilities by a network of signaling pathways where CXCR4/SDF signaling route plays crucial role for directed homing of a polarized cell. The chicken embryo due to its specific vasculature modality has been used as a valuable model for organogenesis, migration, cancer, and metastasis. In this research, the regulatory regions of chicken CXCR4 gene have been characterized in a chicken hematopoietic lymphoblast cell line (MSB1). A region extending from −2000 bp upstream of CXCR4 gene to +68 after its transcriptional start site, in addition to two other mutant fragments were constructed and cloned in a promoter-less reporter vector. Promoter activity was analyzed by quantitative real-time RT-PCR and flow cytometry techniques. Our findings show that the full sequence from −2000 to +68 bp of CXCR4 regulatory region is required for maximum promoter functionality, while the mutant CXCR4 promoter fragments show a partial promoter activity. The chicken CXCR4 promoter validated in this study could be used for characterization of directed migratory cells in chicken development and disease models.

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Acknowledgments

The research work in the laboratory of H.D. is supported by Grant Number 3/25064 from Ferdowsi University of Mashhad, Mashhad, Iran, and Grant Number 100311 from Council for Stem Cell Sciences and Technologies and The Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.

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Authors and Affiliations

  1. Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

    Masoumeh Es-haghi, Mohammadreza Bassami & Hesam Dehghani

  2. Embryonic and Stem Cell Biology and Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

    Hesam Dehghani

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  1. Masoumeh Es-haghi
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Correspondence to Hesam Dehghani.

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Electronic supplementary material

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Supplementary Fig. 1: Sequence alignment of the 3 variant promoter regions of the chicken CXCR4 gene (TIFF 18753 kb)

12033_2016_9917_MOESM2_ESM.tif

Supplementary Fig. 2: Validation of subcloning of the cloned chicken CXCR4 promoter variants. The length of cloned promoter variants was validated by EcoRV and HindIII restriction enzyme double digestion and gel electrophoresis (TIFF 5639 kb)

Supplementary Fig. 3: RT-PCR analysis of the endogenous CXCR4 expression in MSB1 cell line (TIFF 3123 kb)

12033_2016_9917_MOESM4_ESM.tif

Supplementary Fig. 4: Analysis of melting, amplification, and standard curves for the expression of GFP induced by three promoter variants in chicken MSB1 cells. A) The melting curves, B) The amplification curves, and C) The standard curve produced from fivefold serial dilution of positive control cDNA samples (MSB1 cells transfected with CMV-GFP reporter) (TIFF 2401 kb)

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Es-haghi, M., Bassami, M. & Dehghani, H. Construction and Quantitative Validation of Chicken CXCR4 Expression Reporter. Mol Biotechnol 58, 202–211 (2016). https://doi.org/10.1007/s12033-016-9917-2

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