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

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Author: Fernandez, SL
Author: Hurlin, PJ
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Methods Article

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

Sandra L. Fernandez¹, David W. Russell² and Peter J. Hurlin³^*

¹ Shriners Hospitals for Children. Department of Cell Developmental Biology, Oregon Health & Science Portland. OR 97201. USA.
² Department of Pediatrics, Division of Genetics and Developmental Medicine, University of Washington Seattle. WA 98195-7720. USA.
³ Department of Cell Developmental Biology, Oregon Health & Science Portland. OR 97201. USA. Phone: (503) 221 3438 Fax: (503) 221 3451 Email: pjh@shcc.org.

* To whom correspondence should be addressed: Peter J. Hurlin, Department of Cell Developmental Biology, Oregon Health & Science Portland. OR 97201. USA. Phone: (503) 221 3438. Fax: (503) 221 3451. Email: pjh@shcc.org

Biol. Proced. Online 2007;9:84-90. doi:10.1251/bpo136
Submitted: September 10, 2007; Accepted: November 19, 2007; Published: December 24, 2007.

Indexing terms: Dependovirus; Proto-Oncogene proteins c-myc.

Abbreviations: EGFP, enhanced green fluorescent protein; HFF, human foreskin fibroblast; rAAV, recombinant adeno-associated virus.

Figure 2 Enlarged

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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