C-5-13
Site-specific Cellular Modification by Cre-incorporating
Integrase-defective Retroviral Vectors
Takuya Shimomura 1

Shuohao Huang 2

Yoshinori Kawabe 1

2TE12131R@s.kyushu-u.ac.jp

shhuang001@hotmail.com

kawabe@chem-eng.kyushu-u.ac.jp

1

2

Akira Ito 1

Masamichi Kamihira 1,2

akira@chem-eng.kyushu-u.ac.jp

kamihira@chem-eng.kyushu-u.ac.jp

Department of Chemical Engineering, Faculty of Engineering, Kyushu University,
744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku,
Fukuoka 819-0395, Japan

Keywords: Integrase-defective retroviral vectors, Cre/loxP, Site-specific cellular modification
Retroviral vectors have been employed in various biotechnology fields such as gene therapy [1],generation of transgenic animals [2] and induced pluripotent stem cells (iPSCs) [3]. However, thenearly random integration property may cause problems such as insertional mutagenesis and genesilencing. Therefore, the development of targeted transgene integration system is desired formodification of retroviral vectors. We have achieved retroviral insertion of transgene into a
pre-determined site of chromosome in CHO cells using integrase-defective retroviral vectors(IDRVs) and Cre-recombinase mediated cassette exchange (RMCE) [4]. In this case, founder cells
were transfected with a Cre expression plasmid prior to retroviral transduction. To apply
site-specific gene modification for hard-to-transfected cells, it is desired to encapsulate both geneof interest and Cre protein into the retroviral virion. In this study, we attempted to generate novel
hybrid IDRVs, which can deliver both viral genome and enzymatically active Cre.
First, we designed a fusion protein-expression vector (pGPM-Cre), in which a Cre gene wasinserted into the gag-pol precursor genes (pGPM). Cre-incorporating IDRV particles (Cre-IDRV)
were produced by 293FT cells co-transfected using IDRV producing plasmids (pQMSCV/NE,pGPM and pVSV-G) and a pGPM-Cre plasmid. Western blot analysis revealed that Cre wasincorporated into retroviral virions. A Cre-IDRV encoding marker genes (ATG-deleted-Neo r/IRES/EGFP) flanked by wild-type and mutant loxP sites was infected into founder CHO cells(CHO/F17), in which a single copy of an expression unit containing a CMV promoter, an ATG
codon and marker genes (ATG-deleted-Hyg r /IRES/DsRed) flanked by corresponding loxP sites wasintroduced into the genome. We observed appearance of G418-resistant colonies expressing GFP.
Moreover, site-specific integration of transgene was confirmed by PCR and sequence analyses.
These results indicate that bioactive Cre protein was incorporated into virions and site-specificrecombination between retroviral DNA derived from IDRV and cellular genome was mediated byCre-RMCE. The Cre-IDRV system may expand the applicability of retroviral vectors.
[1] Qaism et al., Progress and prospects: Gene therapy for inherited immunodeficiencies. GeneTherapy, 16:1285-1291, 2009.
[2] Kamihira M et al., Production of chimeric monoclonal antibodies by genetically manipulatedchickens. Journal of Biotechnology, 141:18-25, 2009.
[3] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors. Cell, 126:663-676, 2006.
[4] Huang S et al., Cre recombinase-mediated site-specific modification of a cellular genome using
an integrase-defective retroviral vector. Biotechnology and Bioengineering,107:717-729, 2010.