Simultaneous Single Cell Stable Expression of 2-4 cDNAs in HeLaS3 Using .PHI.C31 Integrase System

Abstract

ABSTRACT. An important consideration in the design of multigene delivery technology is the availability of suitable vectors to introduce multiple genes stably and stoichiometrically into living cells and co-express these genes efficiently. As a promising system for this purpose, we developed multi-cDNA expression constructs harboring two to three tandemly situated cDNAs in a single plasmid. The utility of this vector system is amplified by combining it with the φC31 recombinase system which mediates site-specific integration of the genes into naturally occurring chromosomal sequences. By analyzing 55 φC31-mediated integration events with five different constructs, each carrying one, two or three tandem cDNA expression cassettes, we identified 39 pseudo attP sites in the HeLaS3 chromosomes. All these sites share a common motif containing an inverted repeat and showing a similarity to the native φC31 attP. The 36 integration events represented 27 different pseudo attP sites, suggesting the possibility of duplicate integration of the multigene expression plasmids into different genomic loci in a single cell. We demonstrated successive introduction of two different multi-cDNA expression plasmids into definite chromosomal pseudo attP sites, attaining integration of four cDNAs of known genomic constitution at precise genomic loci of a single HeLaS3 cell. The expression levels of these several transgenes were enhanced and made equally stable and robust by inserting the cHS4 insulator between genes.

A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

“…[8][9][10][11][12] Construction of multiple functional DNA elements in tandem on a single plasmid was performed by stepwise LR and BP reactions as indicated in Figure 1b. All functional elements required for Tet regulation were placed on one contiguous DNA fragment of 12 340 bp between att1 and att2 Gateway recombination signals.…”

“…15,16 Unlike Cre and Flp recombinases, fC31 recombinase is a unidirectional enzyme that catalyzes the integration of transgenes into native attachment sequences (pseudoattP) of the host genome of mammalian cells 17 by recombining the attB recognition sequence in a donor plasmid and one or more pseudo-attP site(s) in host chromosomes. [6][7][8] The system described here uses a fC31 attB sequence inserted into the pBKT2 vector backbone (Figure 1a) to enable integration into pseudo attP site(s) of the host chromosomal DNA.…”

“…For this purpose, two full-length cDNAs encoding TetR were cloned downstream from TetO 2 -controlling GOI on a single plasmid (Figure 1), using Multisite Gateway technology [8][9][10][11][12] to assemble 11 DNA fragments. Using a fC31 integrase-mediated recombination system, we constructed stably transfected cells carrying the Tet-inducible expression cassette in a onestep procedure.…”

“…However, the genomic integration sites of plasmids pBKT2-c-Src and pBKT2-Cbp in the transfectants used in this study were at multiple sites. Determination of the insert site by the plasmid rescue and sequencing method 8,30 showed that the pBKT2-c-Src plasmid was inserted at a pseudo attP site positioned 161 bp from XqA1.3, as well asat at least one site by random integration. This insertion was also accompanied by a short chromosomal rearrangement in the flanking DNA sequences downstream from the attRgenome junction.…”

“…Those transgenes introduced into HeLaS3 genomic sites were quite stable and robustly expressed in refreshed culture after longterm storage of over 6 months at À80 1C or in liquid nitrogen (data not shown), consistent with earlier reports. 8,26 By using the fC31 recombination system, we can introduce the all-in-one pBKT2 vector system into essentially any mammalian cell line without pretreatment of cells. We tested the application of this one-step Tet-inducible system to different cell lines such as NIH3T3, Cos7 and 293A, comparing the inducibility with that of HeLaS3 cells.…”