Flp and Cre expressed from Flp-2A-Cre and Flp-IRES-Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange

Abstract

Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35-45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp-2A-Cre and Flp-IRES-Cre transcription units. In contrast, the lowest level of dual RMCE (∼1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp-to-Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes.

Figure 1.

Schematics of dual RMCE (A) and FDL/FEL and LDF/LEF cassettes for testing the efficiency of dual RMCE (B). (A) In the example shown, the vector-borne EGFP-expressing cassette replaces the DsRed-expressing cassette integrated into a genome. At the first, integrative step of the reaction, the incoming vector integrates into one of the compatible recombination targets that flank the DNA fragments being swapped (the incompatible recombination targets, RTa and RTb, are depicted as open and filled triangles, respectively). At the second, deletion step of the reaction, the recombination between the other pair of compatible recombination targets excises the integrated vector and the original genomic DNA fragment leaving the vector-borne one in the genome. (B) FDL/FEL and LDF/LEF cassettes differ from each other by the nature of the recombination targets that flank the promoterless, vector-borne EGFP cassette and the DsRed-expressing cassette integrated into the genome of CHO cells: FRT–loxP or loxP–FRT, respectively. In both cassettes, dual RMCE catalyzed by Flp and Cre replaces the DsRed gene with the EGFP gene. As the result, the EGFP gene can be expressed from the genomic CMV promoter.

Figure 2.

Transcription units that express Flp and Cre. (A) The Flp and Cre expressing cassettes in the (F + C) transcription unit are located on two different vectors; the recombinases are expressed from the CMV promoter. (B) in pDIRE (5), the Flpo and iCre genes are located on the same vector; the recombinases are expressed from the PGK and EF1a promoters, respectively. (C) In the transcription units FIC and CIF, the Flp and Cre genes are located on the same vector and separated by IRES from the pIRES2 series vectors. FIC and CIF are expressed from the CMV promoter. (D) In the transcription units F2AC and C2AF, the coding sequences of Flp and Cre are separated by the 2A peptide TaV (13). F2AC and C2AF are expressed from the CMV promoter.

Figure 3.

Testing dual RMCE efficiency under standard conditions. (A) Typical green colonies formed as a result of LDF × LEF recombination catalyzed by F2AC after the transfected cells were expanded. (B) The PCR analysis confirms that the majority of the green colonies are indeed the result of RMCE and not just simple integration. The green and red bars schematically represent the control PCR products for the replacement and the integration reactions respectively. The control primers anneal upstream of the CMV promoter and downstream of a recombination target; the primers are not specific for the promoter or the reporter genes. The sequencing of the lower, integration-specific band in the variant #6 (LDF × LEF configuration) confirmed the nature of the band. C1, PCR product obtained by subjecting the genomic DNA of the original FDL or LDF cell line, respectively, to the PCR reaction using the control primers. C2, PCR product obtained by subjecting the FEL or LEF reporter, respectively, to the PCR reaction using the control primers. M, 2-log DNA ladder (New England Biolabs). (C) The efficiency of dual RMCE catalyzed by the transcription units F2AC, C2AF, FIC, CIF, pDIRE and (F + C) in the FDL × FEL and LDF × LEF reporter systems is represented by green bars. The concentration of the reporter and the expression plasmids at transfection was 0.4 and 2 µg, respectively. The green bars show the mean value of five experiments; the error bars indicate SD.

Figure 4.

Lowering the concentration of F2AC and pDIRE at transfection differently affects the efficiency of dual RMCE. The results of the LDF × LEF recombination are shown. Lower amounts of F2AC added at transfection lead to the gradual drop in the replacement efficiency. In contrast, the highest efficiency of dual RMCE by pDIRE was achieved by decreasing the concentration of the plasmid at transfection. The concentration of the reporter plasmid at transfection was kept at 0.4 µg; the concentrations of F2AC and pDIRE are indicated. The green bars show the mean value of three experiments; the error bars indicate SD.

Figure 5.

The efficiency of dual RMCE catalyzed by (F + C) is affected by the overall concentration of the recombinase expression vectors and by the relative ratios of the vectors. The results of the LDF × LEF recombination are shown. The concentration of the reporter plasmid at transfection was kept at 0.4 µg; the concentrations of (F + C) are indicated. The green bars show the mean value of three experiments; the error bars indicate SD.