Supplemental Materials

Culture of R1 murine ESCs and their stable transfection with HSV1-sr39tk

Murine ESCs designated as R1 were kindly provided by Dr. A. Nagy’s Laboratory at Mount Sinai Hospital (Toronto, ON, Canada) and were maintained in an undifferentiated state on a feeder layer of inactivated murine embryonic fibroblasts and Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 15% fetal calf serum (Hyclone, Logan, UT), 2mM non-essential amino acids, 2mM L-glutamine, 1xPenn-Strep (all purchased from Invitrogen, Carlsbad, CA), 0.1mM ß-mercaptoethanol (Sigma, St. Louis, MO), and 1000 U/ml recombinant murine leukemia inhibition factor (ESGRO, Chemicon, Temecula, CA).

The 1.2 kb fragment of sr39tk gene was excised from pcDNA-sr39tk, which is kindly provided by Dr. S.S. Gambhir at Stanford University (Palo Alto, CA), and subcloned into the MCS1 and MCS2 sites of pVitro1 (Invivogen, , San Diego, CA) through blunt-cohesive ends ligation. The resulting construct pVitrol-sr39tk carried 2 copies of the sr39tk gene under the control of the promoter of ubiquitous elongation factor 1 alpha (EF-1α) gene from rat and mouse origins respectively. The EF-1α promoter is used because it is capable of driving long-term expression of the sr39tk gene either in differentiated or undifferentiated ES cells (1). It should be noted that when ESCs remain undifferentiated, this promoter is expected to drive the same level of reporter gene expression in each cell, however, when ESCs start to differentiate, the strength of the promoter may fluctuate in different types of cells (see Discussion in the main text).

After linearization using the unique ApaLI restriction site, the plasmid was transfected into R1 cells via electroporation: about 107 cells were suspended in DMEM. Electroporation was performed on a Gene Pulser II system (Biorad, Hercules, CA) using 25v and 500microF(2). After transfection, cells were maintained in cell culture medium containing G418 (250 μg/ml) for 14 days. Thirty G418resistantclones were picked and propagated in the presence of LIF and G418. Levels of HSV1-sr39tk expressionin these clones were examined by tracer uptake studydescribed below.

Uptake of FHBG by HSV1-sr39tk expressing ESCs

To examine whether acycloguanosine derivatives (e.g., 9-[3-Fluoro-1-hydroxy-2-(propoxymethyl]guanine, FHBG) are preferentially taken up by HSV1-sr39tk expressing cells, cells from G418 resistant clones were seeded into 24-well plates in triplicate. After removal of the growth medium, cells were incubated with DMEM containing 0.5 μci/ml [F-18]FHBG for 1 hour at 37°C. Cells were then dissolved by 2N NaOH after extensive washing with PBS. The radioactivity of [F-18] was measured simultaneously from the cell lysate and from the incubation solution on a gamma counter (Cobra II, PerkinElmer life and Analytical Sciences, Boston, MA). Cellular protein per clone was determined using a protein assay kit (Pierce Biotechnology, Rockford, IL). Results were expressed as % uptake per 100µg cellular protein. The clone that has the highest tracer uptake was designated as R1-sr39tk and was used for in vivo studies.

Autoradiography (ARG)

The animals (n=3) were injected with 5million R1-sr39tk cells that were also labeled with super paramagnetic iron oxide particles (3). They wereeuthanized at 3-5h after cell injection and 1h after intravenous (tail vein) injection of 1.5±0.3 mCi [F-18]FHBG. The heart was excised, rinsed with saline and frozen in a dry ice/acetone bath. After reaching equilibrium at -20°C, 30 µm sections were cut on a cryostat microtome (Hacker Instruments, Fairfield, NJ) and thaw-mounted on microscope slides (3 sections per slide). Tissue sections were air-dried at room temperature and exposed to Cronex MRF-34 film for 20 hours. The exposed film was developed by a Kodak automatic film processor. To identify grafted cells, 10 µm sections adjacent to ARGsections were stained for Prussian blue (3).

Design of Taqman probes

Taqman probes (including PCR primers and a probe for minor groove binder,MGB) for detection of the Sry gene were purchased from Applied Biosystems (Austin, TX,Assay ID: m00441712_s1). For an internal standard, an autosomal single copy gene, X-box-binding protein-1 (xbp1) was utilized since it expresses in both mouse and rat tissues of male and female origin. A homology sequence of Xbp1 was carefully identified by the NCI BLAST software to ensure targeting to regions of the Xbp1 gene common to both rat and mouse. The custom made Taqman probes for Xbp1 detection are as follows:

PCR primer forward:5’-CCAGAGGTCTACCCAGAAGGA-3’

PCR primer reverse: 5’-TGATGAGGTCCCCACTGACA-3’

MGB-labeled probe: CCAGCCTCCCTTTCTC.

Each probe has a 5’-FAM dye tag as a reporter and 3’ non-fluorescent quencher. The amplicon length of the Sry and Xbp1 qRT-PCR products is 77bp and 69bp, respectively.The internal standard is necessary because even a small variation in DNA loading could be amplified significantly (by a factor of 2Ct) during PCR.

Reference:

1.Zeng X, Chen J, Sanchez JF, et al. Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation. Stem Cells 2003; 21:647-653.

2.Roach ML, McNeish JD. Methods for the isolation and maintenance of murine embryonic stem cells. Methods Mol Biol 2002; 185:1-16.

3.Zhou R, Thomas DH, Qiao H, et al. In Vivo detection of stem cells grafted in infarcted rat myocardium. J Nucl Med 2005; 46:816-822.