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. SarafovaBiology Department, Davidson College, Davidson, NC 28036

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) 4g/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% CO2

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 efficiencyAKR1G1 Double

Positive Thymoma

LipofectamineTransfection plasmid

Transfect cells with mixture

Incubate in 7.5%

CO 2 at 3

7°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

4e/p-eGFP-silDEAE dextran transfection

4e/p-eGFP-sil and TIPflag(CD8)DEAE dextran transfection

TIPflag(CD8)DEAE dextran transfection

AKR1G1

RLM11

eGFP CD8

4e/p-eGFP-sil lipofection

eGFP

4e/p-eGFP-silDEAE dextran transfection

4e/p-eGFP-sil and TIPflag(CD8)DEAE dextran transfection

TIPflag(CD8)DEAE dextran transfection

AKR1G1

RLM11

eGFP CD8

4e/p-eGFP-sil lipofection

eGFP

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

Am

pr

Making Transfection Plasmids

XbaI and EcoRV double digest, forming 1 blunt, 1 sticky end

XbaIXhoI

4e/p-eGFP-sil

1. XhoI digest

2. Klenow

Pr1

4e/p-eGFP-sil

Pr1XbaI

XbaI digest

pSK

pSK

4e/p-eGFP-sil

Pr1 Quick

Ligat

ion

Amp

rAm

pr

pSK.Cd4Pr1 pSK.Cd4Pr1eGFPPr2

HindIII digest, CIP, CIP Inactivate Ligate

MigRI

eGFP

Am

pr

HindIII digest to excise eGFP

eGFP

pSK.Cd4Pr1

Pr1

Am

pr

pSK.Cd4Pr1eGFP

Pr1

Am

pr

eGFP

Ligate

pSK.Cd4Pr1eGFPPr2

Pr1

Am

pr

Pr2

eGF

P

pNEB206A.Cd4Pr2

Am

pr

Ehlers, M., K. Laule-Kilian, M. Petter, C. Aldrian, B. Grueter, A. Würch, N. Yoshida, T. Watanabe, M. Satake, and V. Steimle. 2003. 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: 3594-3604.

McCready, P., R. Hansen, S. Burke, and J. Sands. 1997. Multiple negative and positive cis-acting elements control the expression of the murine Cd4 gene. Biochim. Biophys. Acta. 1351:181-191

Sawada, S., J.D. Scarborough, N. Killeen, Littman D. 1994. A lineage-specific transcriptional silencer regulates Cd4 gene expression during T lymphocyte development. Cell. 77:917-929.

Wildt, K., G. Sun, B. Grueter, M. Fischer, M. Zamisch, M. Ehlers, and R. Bosselut. 2007. The transcription factor Zbtb7b promotes Cd4 expression by antagonizing Runx-mediated activation of the Cd4 silencer. J. Immunol. 179: 4405-4414.

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 B C G

D E F

Pr2

Quick

Ligatio

n

Sil

Am

pr

eGF

P

4e/p-eGFP-sil