Genomic DNA was extracted from mouse mesenchymal cells using the genomic DNA extraction kit (Bioneer, Korea) according to the protocol.

In order to amplify mouse Oct-4 promoter, three set of primers were designated based on the sequence data for Oct-4 promoter (NCBI data for mouse chromosome 17, region: 21834554-21836862) (Table 1). Oligonucleotide primers were ordered from Metabion Company (Germany). The genomic DNA was used as template for the Polymerase Chain Reaction (PCR)(24). Utilizing Ex taq DNA polymerase (TaKaRa, Japan) two sets of PCR were carried out primary amplifications of the two DNA fragments with the size of 1221 and 1292 bp using F1 and R1 primer set and F2 and R2 primer set, respectively. Both fragments were utilized as templates in the next step for SOE-PCR under the following conditions: initial denaturation at 94°C for 5 min, 25 cycles of 94°C for 1 min, 65°C for 1 min and 72°C for 2 min, and a final extension for 5 min at 72°C. Then, the primary PCR pro-ducts were extracted using gel-extraction kit (Qiagen, Germany) to remove excess primers and templates, and the resulting fragments from the primary PCRs were mixed in a 1:1 molar ratio. Thus, the amount of DNA added to the second PCR (SOE-PCR) was approxi-mately 100 ng. SOE-PCR conditions were similar with those of the primary PCRs, ex-cept that F1 and R2 primers were added to amplify a 2309 bp fragment of DNA. Finally, a nested PCR was performed using F3 and R3 primers with 2 µl the SOE-PCR product as a template.

In order to amplify mouse Oct-4 promoter, three set of primers were designated based on the sequence data for Oct-4 promoter (NCBI data for mouse chromosome 17, region: 21834554-21836862) (Table 1). Oligonucleotide primers were ordered from Metabion Company (Germany). The genomic DNA was used as template for the Polymerase Chain Reaction (PCR)(24). Utilizing Ex taq DNA polymerase (TaKaRa, Japan) two sets of PCR were carried out primary amplifications of the two DNA fragments with the size of 1221 and 1292 bp using F1 and R1 primer set and F2 and R2 primer set, respectively. Both frag-ments were utilized as templates in the next step for SOE-PCR under the following con-ditions: initial denaturation at 94°C for 5 min, 25 cycles of 94°C for 1 min, 65°C for 1 min and 72°C for 2 min, and a final extension for 5 min at 72°C. Then, the primary PCR products were extracted using gel-extraction kit (Qiagen, Germany) to remove excess primers and templates, and the resulting fragments from the primary PCRs were mixed in a 1:1 molar ratio. Thus, the amount of DNA added to the second PCR (SOE-PCR) was approximately 100 ng. SOE-PCR conditions were similar with those of the primary PCRs, except that F1 and R2 primers were added to amplify a 2309 bp fragment of DNA. Finally, a nested PCR was performed using F3 and R3 primers with 2 µl the SOE-PCR product as a template.

Thermal program was carried out based on the following conditions: 1 cycle of 5 min at 95°C as denaturation, 35 cycles of 1 min at 94°C, 1 min at 60°C and 2 min at 72°C and 1 final cycle of 15 min. at 72°C. Amplicon with expected size (2177 bp) was purified by gel extraction kit (Qiagen, Germany). The purified product was then cloned in a T/A cloning vector using T/A cloning kit and pTZ57/R cloning vector (Fermentas) according to the manual of the manufacturer. DH5α competent cells were transformed by the product of the T/A cloning reaction and colony selection was performed by white and blue screening method on LB plates containing ampicillin (100 µg/ml), IPTG and X-gal. This vector was named pTZ57/Oct-4 promoter plasmid.

A mammalian expression vehicle (25) including pCMV-Int and pDB2, encoding PhiC31 integrase and EGFP coding region flanked by the cytomegalovirus promoter respectively, was kindly provided by Prof. Calos (Stanford University, USA).

In order to construct a recombinant pDB2 plasmid expressing EGFP under the control of Oct4 promoter, a fragment encompassing the sequences of CMV promoter in pDB2 was removed by double digestion with NheI and VspI (Fermentas, Lithuania). Subsequently, the recombinant pTZ57/Oct-4 promoter plasmid was double digested with the same restricted enzymes. To obtain Oct-4 promoter/EGFP cassette, the ligation was performed with digested pDB2 and the fragment containing Oct4 promoter using DNA Ligation Kit (Takara, Japan). Similarly, a plasmid without the CMV promoter (▵ promoter/EGFP) was constructed using digested pDB2 plasmid which its ends were blunted by klenow and ligation reactions. This construct was used as a negative control.

Again, DH5α competent cells were transformed by the ligation mixture and positive colonies were screened on LB plates containing kanamycin (30 µg/ml). Colony PCR was performed using F1 and R1 primers to select bacterial colonies with the recombinant plasmids. Selected positive colonies were cultured overnight and recombinant plasmids were purified by Miniprep Kit (Qiagen) and sequenced (Metabion company, Germany) to ensure the accurate cloning of Oct4 promoter without any mutation.

Royan B1, mESCs derived from the C57BL/ 6 strain of mouse (26) were grown on a feeder layer of the primary Mouse Embryonic Fibroblasts (MEF) in tissue culture flasks. Cells were maintained in ESCs medium that consisted of DMEM (Gibco) containing 15% fetal bovine serum (Gibco), 0.1 mM β-mercap-toethanol (Sigma), 2 mM glutamine (Gibco), 0.1 mM non-essential amino acids (Sigma) and 1000 IU/ml leukemia inhibitory factor (Chemicon).

Transient transfection of mESCs was performed by electroporation. Approximately 3.5×10⁶ of mESCs were re-suspended in PBS buffer (250 µl) and mixed with the recombinant plasmids, including Oct-4 promoter/ EGFP (10 µg) and negative control. Moreover, pCMVInt (10 µg) was added to each tube to perform co-transfection. Each cell/DNAs mixture was transferred into an electroporation cuvette (4 mm gap) and pulsed in the gene pulser using the following conditions: U=280 V, C=500 µF.

After electroporation, mESCs were allowed to spread on pre-coated gelatin-plastic flasks containing mitomycin C-inactivated feeder layer of primary cultures of mouse embryonic fibroblasts (MEF) in ESC medium containing G418 (Sigma, USA). Cell culture incubation was performed for 10 days in a humidified atmosphere of 5% CO₂ at 37°C. Finally, the preliminary screening was carried out for pDB2/Oct-4 promoter by PCR.

Two independent G418-resistant colonies were picked up, cultured and routinely passaged every 2 days in ESCs medium containing 100 µM G418 (Invitrogen). After 4 passages, G418-resistant ESCs were obtained and grown on a feeder layer of primary mouse embryonic fibroblasts. Genomic DNA of the stable cell lines were extracted using genomic DNA extraction kit and the presence of the EGFP was shown in both Oct4 promotor/ EGFP and ▵ prompter/EGFP by PCR.

The percentage of GFP expressing cells and fluorescence intensity was assessed by flow cytometry. Control cells, ▵ promoter/ EGFP cells, were used to determine the average number of GFP-positive cells. For the flow cytometry analysis, the ESCs were isolated from MEFs by treatment of the cells with trypsin-ethylenediamine tetraacetic acid (EDT A) solution (Gibco). The supernatant containing cells was then collected and centrifuged (6 min, 1,200 rpm). The isolated cells were resuspended (5×10⁵ cells/ml in phosphate buffered saline (PBS) and assessed by a flow cytometer (Becton Dickinson) at 488 nm. Acquired data were then analyzed using Win MDI software.

To obtain the sequence of the basic elements of Oct-4 promoter, several sets of PCR on the basis of the DNA sequence of mouse mesenchymal cells were carried out using designated primer pairs (Figures 1A and B). Several fragments were obtained by SOE-PCR. Fragment 1 consists of 1221 bp (Figure 1C, left hand panel, Frag 1) which corresponds to the upstream part of the Oct-4 promoter. Fragment 2 contains 1292 bp, which includes both repeated motif (CCC A/T CCC) and putative consensus sequence for Sp1 (Figure 1C, left hand panel, Frag 2). Finally, the full-length of Oct-4 promoter was amplified by nested PCR using cloning primers. Amplicon was approximately 2.17 kb and it was extracted from agarose gel. Then it was inserted into the T/A vector and was sequenced (Figure 1C, right hand panel, Frag 3). The sequence has shown 100% identity to Oct-4 promoter sequence (GenBank accession no. NT_039649.7). Finally, cloning of the amplicon was achieved by insertion into pDB2 vector in place of CMV promoter. The recombinant vector was named pDB2-Oct4 (Figure 2A).

Bacterial colony insert check on the transformed colonies was evident that two independent colonies out of ten colonies contained the recombinant vector, pDB2-Oct4 (Figure 2B). This recombinant expression vector encoding the EGFP reporter gene in conjunction with Oct-4 transcriptional response element was a reporter cassette user extracted and used for the next experiment.

In order to develop an Oct-4 based cellular reporter line as a pluripotency marker, mESCs, Royan/B1cells were transfected with aforementioned reporter cassette using a non-viral strategy, which a reporter embryonic cell line was created. Transfection was carried out using both user pCMVInt and the reporter construct, and the transient cells were obtained with high efficiency (data not shown). Cells were screened by PCR and RT-PCR to confirm the presence of GFP (data not shown). Positive colonies were passaged four times in the presence of G418 to generate stably transfected cell line. The results showed that reporter cassette was able to express both EGFP and G418 resistance coding sequences for selection in mouse ES cells.

To confirm that the new reporter from mESCs is functional, live cell imaging was performed. Both the stable mESCs and control cell lines were grown in pre-coated gelatin-plastic flasks containing feeder layer of neomycin resistance MEFs in ESC medium which were utilized at 80-90% of confluency. The ability of the new cell reporter to produce detectable signal was assayed using the fluorescence microscopy. Interestingly, large number of cellular reporter expressed EGFP under the control of Oct4, and were imaged under fluorescence microscopy as shown in Figure 3A. The results indicated that the screened Oct-4 promoter/EGFP cell line was the clonal population that exhibits the strongest expression in comparison to the control and interestingly retains the embryonic features of the parental Royan /B1 cells.

The quantitative studies of the new Oct-4 based gene reporter was carried out using the flow cytometry. Both the new cell reporter and control cell [(stably transformed with pDB2 (▵ promoter/EGFP) plasmid)] lines were treated by trypsin and assayed using a FACS with excitation at 488 nm for EGFP. The results were collected at 530/30 nm and the expression efficiency of EGFP in Royan/ B1 cell was analyzed using WinMDI 2.9 software. As shown in Figure 3B, the stably cellular reporter line that constitutively expressed EGFP could be detected with strong signal (up to 95%) by flow cytometry (Figure 3B). The results were consistent with the results obtained from fluorescence microscopy which serves as a qualitative index of Oct4 activity, and it seems that the new reporter system-Royan B1 Oct4/EGFP-allows analyzing the effect of differentiation-prompting agents with high efficiency.