Characterization of a positive transcriptional control element in the Cd4 gene in mice using transient transfection of double positive thymoma cells Darina Spasova and Sophia D. Sarafova Biology Department, Davidson College, Davidson, NC Abstract Methods Acknowledgments References A B C Figure 1 : AKR1G1 double positive thymoma cells successfully transfected with 4e/p-eGFP-sil fluoresce green (A). Transfection efficiency using the DEAE dextran method is low, regardless of the amount of DNA (B) 4  g/mL, (C) 6  g/mL, or the time allowed for recovery and eGFP expression after transfection - 24h (B) vs. 48h (C) Higher level of eGFP expression was observed at the later time points. Figure 2: Flow cytometry data from a DEAE dextran transient transfection of AKR1G1 and RLM11 double positive thymoma cell lines, using 4e/p-eGFP-sil as the experimental transfection plasmid and TIPflag(CD8) as the transfection control plasmid (A, B, C, D, E, F). Lipofection of AKR1G1 double positive thymoma cell line using 4e/p-eGFP-sil as experimental transfection plasmid and no plasmid as experimental control (G). A flow cytometry assay was performed on the double transfection using the eGFP fluorescence, which the transfection plasmid was supposed to introduce into the cells (A, D). The single transfections were not stained (B, E). Cells were stained with CD8FITC when transfection efficiency was to be tested for the control plasmid - TIPflag(CD8) (C, F). Even though they were visible under a fluorescent microscope, AKR1G1 and RLM11 transfected cells with 4e/p-eGFP-sil were not detectable by FACS (B, E). CD8 transfection is readily visible by FACS in the AKR1G1 cell line (C) but not in the RLM11 cells (F). Blue line represents no plasmid transfection. Red line represents plasmid transfection (stained with CD8-FITC in C, F). Black line represents no plasmid transfection stained with CD8-FITC (stained with CD8-FITC in C, F). Transient Transfection using DEAE dextran Transfection plasmid RLM-11 Double Positive Thymoma AKR1G1 Double Positive Thymoma DEAE Dextran Transfection plasmid Incubate in 7.5% CO 2 at 37°C 24, 48, 72 hour time points Fluorescent Microscope Examination and FACS analysis to check for transfection success and efficiency Transient Transfection using Lipofectamine Fluorescent Microscope Examination and FACS analysis to check for transfection success and efficiency AKR1G1 Double Positive Thymoma Lipofectamine Transfection plasmid Transfect cells with mixture Incubate in 7.5% CO 2 at 37°C DEAE dextran and lipofection are not suitable for transfection of DNA into AKR1G1 DP thymoma cells Low efficiency of DEAE dextran transfection of DNA into AKR1G1 DP thymoma cells Conclusions and Future Directions  DEAE dextran and lipofection are both not highly efficient techniques for transfecting DNA into AKR1G1 double positive thymoma cells.  More protocols need to be examined in order to achieve high efficiency of transfection. One possibility is using an electroporator.  When a high efficency protocol is selected, the newly made transfection plasmids will be able to demonstrate whether the transcriptional control element (Pr2) is an enhancer or a promoter. pSK.Cd4Pr1 Pr1 Amp r Making Transfection Plasmids XbaI and EcoRV double digest, forming 1 blunt, 1 sticky end XbaI XhoI 4e/p- eGFP-sil 1.XhoI digest 2.Klenow Pr1 4e/p- eGFP-sil Pr1 XbaI XbaI digest pSK 4e/p-eGFP- sil Pr1 Quick Ligation Amp r pSK.Cd4Pr1pSK.Cd4Pr1eGFPPr2 HindIII digest, CIP, CIP Inactivate Ligate MigRI eGFP Amp r HindIII digest to excise eGFP eGFP pSK.Cd4Pr1 Pr1 Amp r pSK.Cd4Pr1eGFP Pr1 Amp r eGFP Ligate pSK.Cd4Pr1eGFP Pr2 Pr1 Amp r Pr2 eGFP pNEB206A.Cd4Pr2 Amp r Ehlers, M., K. Laule-Kilian, M. Petter, C. Aldrian, B. Grueter, A. Würch, N. Yoshida, T. Watanabe, M. Satake, and V. Steimle Morpholino antisense oligonucleotide-mediated gene knockdown during thymocyte development reveals role of Runx3 transcription factor in Cd4 silencing during development of Cd4 - CD8 + thymocytes. J. Immunol. 171: McCready, P., R. Hansen, S. Burke, and J. Sands Multiple negative and positive cis-acting elements control the expression of the murine Cd4 gene. Biochim. Biophys. Acta. 1351: Sawada, S., J.D. Scarborough, N. Killeen, Littman D A lineage-specific transcriptional silencer regulates Cd4 gene expression during T lymphocyte development. Cell. 77: Wildt, K., G. Sun, B. Grueter, M. Fischer, M. Zamisch, M. Ehlers, and R. Bosselut The transcription factor Zbtb7b promotes Cd4 expression by antagonizing Runx-mediated activation of the Cd4 silencer. J. Immunol. 179: Regulation of Cd4 gene expression cannot be fully explained by the transcriptional control elements described to date. Previous research in the lab revealed that there are two different transcripts present in developing and in mature T cells, which, we speculated, showed the presence of another positive control element within intron 1 of the Cd4 gene. In this study, we describe an in vitro approach to determine if this positive regulatory element is a promoter or an enhancer. We cloned a piece of intron 1, immediately upstream of exon 2, and wished to examine its promoter and enhancer capabilities in a transient transfection assay. For that purpose, we started constructing plasmids in which we placed the DNA of interest in front of a reporter gene (eGFP) in the presence or absence of other known Cd4 transcriptional control elements. Furthermore, we attempted to establish a high efficiency transient transfection protocol, such that successfully transfected cells can be visualized by flow cytometry. Neither of the two different protocols gave us high efficiency transfection. More protocols will be tested in the future, while the reporter plasmid construction is finished. We would like to thank Chris Healey for providing us with technical support, the NIH – for all the reagents they donated to our lab, Karmella Haynes for the insight and reagents for lipofection, and Dr. Yuan Zhuang at Duke University Medical Center for providing us with transfection and control plasmids. A BCG DEF Pr2 Quick Ligation Sil Amp r Pr1 eGFP 4e/p- eGFP-sil