Viral Cre-LoxP tools aid genome engineering in mammalian cells. Issue 1 (December 2017)

• Record Type: Journal Article
• Authors: Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao
• Is Part Of: Journal of biological engineering. Volume 11:Issue 1(2017)
• Page Start: 1
• Page End: 9
• Publication Date: 2017-12
• Subjects: Cre-LoxP -- Cre recombinase -- Lentiviral vector -- AAV vector -- Genome editing -- Talen; Bioengineering -- Periodicals; 660.6
• DOI: 10.1186/s13036-017-0087-y
• Languages: English
• ISSNs: 1754-1611

Abstract

Abstract Background Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. Results We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. Conclusions We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.