1. Introduction 1.The mouse T-cell receptor α -chain (TCR α ) gene is expressed in incredibly reliable and predictable patterns (Table 1), which is a highly desirable characteristic for modern day gene therapy (Johnson et al. 2009; Lahiji et al. 2013). 2. The mouse TCR α /TCRδ/Dad1 gene locus is thought to be responsible for these predictable expression patterns (Diaz, Cado and Winoto 1994). 3. Scientists have attempted to mimic the effects of this locus by developing bacterial artificial chromosomes (BACs) that include elements from it. These BACs are then transfected into reporter mouse models (Harrow and Ortiz 2005). 4. There has been some success in recreating the patterns in vivo, however several of them are still unaccounted for. It is presumed that other gene regulatory elements of the endogenous TCR α locus are responsible for these missing characteristics. The lab I worked in attempted a new BAC. Copycat – or mouse: An Attempt to Mimic Mouse T-Cell Receptor Alpha (TCRα) Chain Expression Patterns Conor Murphy, Martina Kucerova-Levisohn, Dr. Benjamin Ortiz Department of Biology, York College of Pennsylvania Hunter College SPUR Objectives  Determine baseline levels of hCD2 reporter gene expression in the thymus of the transgenic mice  Determine the hCD2 reporter gene’s onset of expression during T-cell development in the transgenic mice Experimental BAC TCRα Onset of expression between DN3  DN4  T-cell specific  High levels of expression in thymus  Upregulation in peripheral organs  Table 1. List of predictable patterns exhibited by the expression of mouse TCR α, and whether or not they were present in the previous experimental mouse model. I would like to thank Hunter College CUNY of New York, New York and their summer research program for this opportunity. I also want to thank Martina Kucerova-Levisohn and Dr. Benjamin Ortiz for their continuous support and mentorship throughout this research project. Supported by NIH-RCMI grant numbers MD007599 (formerly RR003037) and NIDA grant R25DA032520. Extract thymus Enrich for DN T cells (<10% of T cells) Stain DN T cells with antibodies (CD44, CD25, hCD2) Run cells through a flow cytometer NTGWT BAC62WT BAC71 Conclusions Results Figure 1. The TCR α LCR genomic region and reporter transgenes. (A) Diagram of the TCRα/Dad1 locus. (B) Depiction of the hCD2 and hCD2:1-8 transgenes. Figure 2. Upregulation of the hCD2:1-8 reporter gene at DN3 or during the DN3 to DP stage transition. Flow cytometric analysis of two representative hCD2:1-8 clones. Future Directions Literature Cited  Determine the reason for early upregulation  Try a different promoter  Test the BAC for additional expected patterns:  High hCD2 expression in peripheral T cells  Cell type-specific expression  Attempt transfection in vitro, perhaps with mouse embryonic stem cells (ESCs) Diaz, P., Cado, D., and Winoto, A. (1994). A locus control region in the T cell receptor alpha/delta locus. Immunity 1: 207-217. Harrow F, Ortiz BD. (2005) The TCR-alpha locus control region specifies thymic, but not peripheral, patterns of TCRalpha gene expression. J. Immunol. 175:6659-67. Johnson L. A., Morgan R. A., Dudley M. E., Cassard L., Yang J. C., Hughes M. S., et al. (2009).Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114, 535–546.10.1182/blood-2009-03-211714 Lahiji A, Kucerová-Levisohn M, Lovett J, Holmes R, Zúñiga-Pflücker JC and Ortiz BD. (2013) Complete TCR-alpha gene locus control region activity in T cells derivedin vitro from embryonic stem cells. J. Immunol. 191:472-9. Methods Acknowledgements 1.There was a clear positive shift in hCD2 expression patterns. Thus, the combination of elements included into the newly designed BAC are able to recreate this characteristic. 2.There was a drastic upregulation of hCD2 expression sometime between the DN3 and DN4 stages of T cell development. This also shows that the elements present are able to recreate this characteristic. 3.The original onset of expression began far too early as compared to the endogenous expression patterns. We observed an upregulation of hCD2 expression sometime before the DN1 stage of T cell development. 4.The newest BAC for this type of study, therefore, does not fully support both of the predicted expression patterns that I studied – onset of expression between DN3  DN4, and high levels of expression in the thymus.