Contribution

In our project, we utilized the Sleeping Beauty（SB）transpose system to generate stable HEK-293T cell lines. Herein, two parts from the registry were used to generate the donor plasmid. The Sleeping Beauty 5' ITR ,(BBa_K426000) ;part was submitted by team iGEM10_Berkeley and encodes the 5’ transposase recognition site of the sleeping beauty system. The part encoding Sleeping Beauty 3' ITR (BBa_K426009) ;was also submitted by the same team, which provided the 3’ transposase recognition site of the sleeping beauty system. However, we found important information missing on the registry pages of these parts. Therefore, we made efforts to supplement background information and provided experimental validation data and our protocol to generate stable cell lines with the Sleeping Beauty system.

The sleeping beauty system is a synthetic transposon that was constructed based on sequences of transpositionally inactive elements isolated from fish genomes. It is a Tc1/mariner superfamily transposon allowing a cut-and-paste transpositional reaction (Ivics et al., 2004) . Compared with the classical lentivirus-mediated transgenic methods, SB-mediated transgenesis is less prone to gene mosaicism and gene silencing. Transposon vectors can be combined with specific promoter elements, which can control the gene expression levels in tissues and therefore specifically control the expression of transgenes.

The SB vector is composed of two functional components, an SB transposase, and a donor DNA carrying the transposon. These two components are usually encoded separately by two different plasmids: a donor plasmid carrying the target gene flanked by 5’- and 3’-ITRs, and another SB transposase-expressing plasmid. Once co-transfected into the cells, the SB transposase will recognize the ITR sequence and transfers the gene from the donor DNA to the recipient site within the genome. In specific, when the transposase recognizes and binds to the ITR sequences flanking the transposon, they bring the ITRs together to form a circular synaptic complex. In this complex, both ends of the transposon are paired and held together by transposase subunits. Following the assembly of the synaptic complex, the excision of the SB transposon from the donor locus occurs and the integration into the host genome follows (Ochmann & Ivics, 2021).

Figure1. Schematic drawing of Sleeping Beauty transposition. (a) The SB transpose binds to the DRs within the TIRs. (b) The TIRs are brought together by SB transposase molecules in the synaptic complex. (c) The excised transposon integrates into a TA site in the target DNA

To generate stable cell lines producing both green and red fluorescent signals, we first generated two SB donor plasmids carrying either a P_RPBSA-dtomato-2A-BlastR or a P_RPBSA-EGFP-2A-PuroR transposon. HEK-293T cells were co-transfected with both donor plasmids and the transposes expressing P_hCMV-SB100X plasmid in a 10:10:1 ratio. Cells were treated with Blasticidin (10 µg/mL) and puromycin (0.5 µg/mL) since the 8^th h post transfection.

Fluorescent imaging of the stable cell line shows a unanimous expression of EGFP and dTomato. A high heterogeneity in regards of the EGFP/dTomato ratio among the stably transfected cells can also be observed.

Figure2. Fluorescence imaging of stable HEKstress cells