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Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice
 
Molishree U. Joshi1,2, H. Keith Pittman2, Carl E. Haisch2, and Kathryn M. Verbanac1,2
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Metastatic Tumor Model and Intravenous EGFP-VEC Injection

Lewis lung adenocarcinoma (LLCaB) cells were derived from an LLC1 (CRL-1642; ATCC, Manassas, VA, USA) metastatic tumor and cultured as described (18). The metastatic model was originally reported by Folkman (19) and modified by our laboratory (20). Mice bearing lung and liver metastases were injected intravenously with 2 × 106 EGFP-VEC and euthanized 24 h later. Organs were flash frozen for transgene copy number determination or frozen in Tissue Tek-OCT (optimum cutting temperature) compound (Sakura Finetek, Torrance, CA, USA) for biolocalization studies. Strict measures were taken to avoid sample cross-contamination. Separate surgical instruments were used for each mouse.

The control groups in this study were: (i) healthy mice not injected with EGFP-VEC (n = 5); (ii) metastases-bearing mice not injected with EGFP-VEC (n = 4); and (iii) healthy mice injected with EGFP-VEC (n = 5). The tumor group comprised metastases-bearing animals injected with EGFP-VEC (n = 6).

Subcutaneous Tumor Model and Intratumor EGFP-VEC Injection

Mice were injected subcutaneously (s.c.) in one flank with 5 × 105 LLCaB cells. Resulting tumors (∼150 mm3) were injected with 50 µl saline (n = 4) or 2 × 105 EGFP-VEC in 50 µl media (n = 9). Mice were euthanized 2 h post-injection and tumors were frozen in OCT. Alternating 10 µm sections were examined by fluorescence microscopy or extracted for DNA. One mouse was removed from further analysis as it could not be enumerated by microscopy due to high tissue autofluorescence.

Confocal Microscopy

Four sections per s.c. tumor were randomly selected and corresponding z-stacks containing 10 1-µm optical sections were generated (Model no. LSM510; Carl Zeiss Micro Imaging, Thornwood, NY, USA). Four 100 µm2 areas per tumor, each containing an apparent fluorescent cell, were similarly analyzed. The average and maximum fluorescent intensities were measured.

Isolation of Genomic DNA from Tissue

Genomic DNA was extracted from murine liver and lung tissue sections by the Phenol-Chloroform method (21) with the following modifications: (i) thawed 10–50 µm tissue sections were rinsed with PBS (320 osm, pH 7); (ii) an additional 1-h incubation with RNase (0.025 mg/mL) was included after tissue digestion in lysis buffer; and (iii) an additional rinse with chloroform/isoamyl alcohol was included after Genomic DNA extraction with phenol-chloroform-isoamyl alcohol. DNA concentration was always determined with a fluorescence assay using SYBR-green dye (22), which was more accurate than spectrophotometric determination (OD260).

Plasmid

pEGFP-N3 was a gift from Alexander Murashov (East Carolina University). DH5α cells were transformed with pEGFP-N3 by standard methods. Plasmids were purified using the Plasmid Mega kit (Qiagen, Valencia, CA, USA).

The mass of one copy (or molecule) of the plasmid pEGFP-N3 was calculated from the following formula:



m = mass of one copy = M/NA where



M = the molecular weight (Dalton or gram/mole) of the plasmid (calculated using the base composition of the plasmid and MW of nucleotides) = 3.09 × 106 and



NA = Avogadro's number = 6.02 × 1023 copies (or molecules)/mole.

Thus, we calculated that each molecule, or copy, of pEGFP-N3 has a mass of ∼5.1 attograms (ag), and 1 ng of pEGFP-N3 has approximately 1.95 × 108 copies of EGFP. The number of plasmid copies per microliter was determined by dividing the concentration of plasmid by mass (m). A pEGFP-N3 plasmid standard curve was constructed in each assay, as described in Figure 1.





qPCR

qPCR was conducted with 100 ng DNA in duplicate in a 25 µl reaction using the TaqMan PCR Universal Master Mix and the Applied Biosystems 7000 Real-Time System (both from Applied Biosystems, Foster City, CA, USA). Primer and probe sequences for EGFP were from Jackson Laboratories (personal communication) and manufactured by Applied Biosystems: forward primer 5′-ccacatgaagcagcaggactt-3′, reverse primer 5′-ggtgcgctcctggacgta-3′ and probe 5′-6FAM-ttcaagtccgccatgcccgaa- TAMRA-3′. The final concentration of EGFP primers was 400 nM each, and the probe was 150 nM. Gene Expression Assay Mm00607939_s1 (Applied Biosystems) was used for assaying β-actin (endogenous control) in separate reactions. This proprietary assay resulted in a 115 bp product within exon 6, detected with a FAM/MGB-labeled probe. All reactions were 50 cycles using standard ABI cycling conditions (initial 2 m at 50°C, 10 m at 95°C, and 50 cycles of 15 s denaturation at 95°C and 1 m annealing and extension at 60°C).

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