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. 2007 Dec 24:9:84-90.
doi: 10.1251/bpo136.

Development of human gene reporter cell lines using rAAV mediated homologous recombination

Sandra L Fernandez  1 , David W Russell, Peter J Hurlin
Affiliations

Development of human gene reporter cell lines using rAAV mediated homologous recombination

Sandra L Fernandez et al. Biol Proced Online. .

Abstract

Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used for selection of correctly targeted alleles by taking advantage of known regulatory conditions that activate transcription of specific genes. Using this method, we describe the generation of primary human fibroblasts that express EGFP-Luciferase under the control of the c-Myc oncogene.

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Figures

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Fig. 1
Direct selection of targeted EGFP-Luciferase reporter gene insertion into the c-Myc locus using known c-Myc regulatory conditions. (A) Subconfluent primary human foreskin fibroblast cells are infected with rAAV targeting vectors when c-Myc is being actively transcribed or replicated in proliferative cells. Background reporter gene expression is silenced through density arrest and serum withdrawal. Reporter gene expression is induced by serum stimulation. Single EGFP-Luciferase positive cells are selected using FACS. Cloned cells are expanded and screened for gene targeting events by PCR, Southern blot, and sequencing. (B) FACS histograms are shown for wildtype and c-Myc rAAV targeted cells following cell cycle entry. FL1-A (EGFP) was plotted against FL2-A (no fluorofor) to center the parent population and allow for selection of the dim EGFP-Luciferase positive cells. The P2 region of the plot indicates the gating used for selection of the EGFP-Luciferase positive portion of the parent population.
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Fig. 2
Genotype analysis of EGFP-Luciferase positive clonal populations. (A) Diagram of the c-Myc rAAV targeting and genotype strategies. The targeting region of the endogenous c-Myc locus is shown. The left and right homologous arms (LHA & RHA), and viral inverted terminal repeat portions of the rAAV vectors facilitate insertion of the EGFP-Luciferase fusion gene directly between the first and second codon of the c-Myc gene. K, KpnI. X, XbaI. C, ClaI. S, SspI. (B) Triple primer PCR genotyping was performed using primers 1-3 shown in (A). (C) Southern blot analysis using the c-Myc exon 3 probe shown in (A). (D) Sequence data showing the regions spanning the c-Myc rAAV insertion sites and the internal EGFP-Luciferase insertion regions.
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Fig. 3
MR1 and MR2 response to cell cycle entry. MR1 (A) and MR2 (B) clonal populations were subjected to cell cycle entry conditions and then evaluated for EGFP-Luc reporter gene expression. The unstimulated background control populations are shown in the top panels. Serum stimulated populations are shown in the bottom panels where the EGFP-Luciferase expressing cells are located in the P2 gated region.
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Fig. 4
Comparison of c-Myc and EGFP-Luciferase reporter gene expression and activity kinetics in response to cell cycle entry. The MR1 cells were tracked through CCE for their ability to induced c-Myc and EGFP-Luciferase transcripts by RT-PCR (A) and protein by Western blot (B). Max protein expression is used as a loading control. (C) Luciferase activity of the MR1 population over CCE. Data shown is the average of triplicate samples +/- SEM. (D) FACS Calibur data is shown where the EGFP-Luciferase positive percent of the parent population was determined using BD CellQuest Pro v. 5.2. Time course changes of the percent EGFP-Luciferase expressing populations were compared under the same gates.
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