Site-Specific Recombinase Strategy to Create Induced Pluripotent Stem Cells Efficiently with Plasmid DNA

Abstract

Induced pluripotent stem cells (iPSC) have revolutionized the stem cell field. iPSC are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSC have been developed, but the non-integrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here we report a facile strategy to create murine iPSC that utilizes plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSC. Cre recombinase was then employed for excision of the reprogramming genes. The iPSC were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable to retroviral approaches in efficiency, but is non-hazardous for the user, simple to perform, and results in non-random integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.

Site-Specific Recombination Using PhiC31 Integrase

“…Integration site analysis revealed that 37% of the MEF-derived iPSC clones and 31% of the AD-MSC-derived iPSC clones contained a single integration event. More than three integration events per single clone were not observed (Karow et al 2011 ) . One clone from each population of single integrants was chosen for additional analysis.…”

“…In another recent study, these issues were addressed. The donor plasmid employed contained the same four reprogramming factors used by Ye and colleagues, with the following differences: the f C31 attB site was fl anked by loxP sites, the plasmid additionally contained an R4 integrase attP site, and the tetracycline inducible system was absent (Karow et al 2011 ) . MEFs and mouse adipose-derived mesenchymal stem cells (AD-MSC) were reprogrammed by nucleofection with the donor plasmid and an integrase-expressing plasmid.…”

“…MEFs and mouse adipose-derived mesenchymal stem cells (AD-MSC) were reprogrammed by nucleofection with the donor plasmid and an integrase-expressing plasmid. iPSC-like colonies were obtained, expanded in culture, and integration site analysis was carried out via a combination of Southern blot and linker-mediated polymerase chain reaction (Karow et al 2011 ) . Integration site analysis revealed that 37% of the MEF-derived iPSC clones and 31% of the AD-MSC-derived iPSC clones contained a single integration event.…”

Generation and Genetic Engineering of Human Induced Pluripotent Stem Cells Using Designed Zinc Finger Nucleases

“…Generation of iPSCs from human and murine cells using other similar methods such as piggybac transposons, phage integrase (FC31), and excisable lentiviruses have been reported elsewhere [32,33]. Although the FC31-derived hiPSCs had only a single integration in each line, the locations of integration were random in different lines, favoring intergenic regions [32].…”

Pluripotent stem cells to hepatocytes, the journey so far

“…Many studies have explored the removal of viral vectors after reprogramming. These techniques include plasmid transfection and piggyback transposition system, in which the inserted transgene can be subsequently excised to remove the risk of residual expression and re-activation of reprogramming factors (18,19). The risk of insertional mutagenesis still remains, as the sequences beyond the Lox site cannot be fully excised.…”