CRO Service

Transposon-based Pathway Integration

Stably integrate multi-gene pathways into mammalian host genomes without viral vectors, accelerating cell line development for bioproduction and cell-based assays.

PiggyBac or Sleeping Beauty transposon platform
Customizable promoter and gene order design
Stable pools or clonal cell lines delivered a timeline confirmed during project.

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Rapid Stable Integration Without Viral Vectors

Our transposon-based service enables stable expression of large or complex pathways (metabolic, signaling, reporter) in CHO, HEK293, or other adherent/suspension mammalian cells. The process is fully scalable from research to pilot production.

Tailored vector construction with ITR-flanked cassettes
Co-transfection with transposase (plasmid or mRNA)
Selection, single-cell cloning, and copy-number/expression QC

CHO Host

HEK293 Host

Custom Promoters

Approach

Stable Integration of Multi-Gene Pathways via Engineered Transposons

This service uses a non-viral transposon system (PiggyBac or Sleeping Beauty) to integrate your pathway of interest (up to 100+ kb) into the host genome. The transposon vector carries the pathway genes flanked by inverted terminal repeats (ITRs); co-delivery of transposase excises the cassette from the plasmid and inserts it into genomic TTAA (PiggyBac) or TA (Sleeping Beauty) sites. Integration is stable across generations without the need for continued selection, and copy number can be roughly controlled through transposase/vector ratios. We handle all steps from vector design to final cell bank generation.

Custom Vector Design and Construction

We design and synthesize the transposon vector to your pathway specifications. Choose promoter combinations (CMV, EF1a, CAG, etc.), orientation, and polycistronic configurations (IRES, 2A), and we will insert the fully assembled cassette flanked by ITRs into the backbone.

Multiple promoters per cassette for differential expression
IRES or 2A peptide for multi-cistronic expression
Optional fluorescent or selection markers

Transfection and Stable Pool/Clone Generation

We co-transfect the transposon vector with transposase (as plasmid or mRNA) into the target cell line. After antibiotic selection or FACS sorting, we expand stable pools or perform limiting-dilution cloning to isolate isogenic clones.

Selection markers (puromycin, blasticidin, hygromycin, etc.)
Single-cell cloning with imaging or automated picking
Cryopreserved research cell banks available

Non-Viral, Low Immunogenicity

No viral components are used; integration relies solely on the transposase enzyme, reducing safety concerns and regulatory burden for later stages.

Large Cargo Capacity

Transposons can accommodate very large DNA inserts (size as scoped per project), enabling the integration of entire metabolic or signaling pathways in a single event.

Stable Long-Term Expression

Integrated transposons persist in the genome after selection is removed, providing stable expression over many passages suitable for production cell lines.

Deliverables

What You Receive

Every transposon engineering project is scoped to your needs. Typical deliverables include:

Cell Line Deliverables

Stable pool or clonal cell line (≥10 vials per clone)
Research cell bank (RCB) cryovials with project documentation
Glycerol stock of the final transposon vector
Expanded parent control cells (when requested)

QC and Documentation

Integration copy number by qPCR (target + reference gene)
Transgene mRNA expression by RT-qPCR (pool and clone)
Sanger or NGS confirmation of ITR-to-insert junctions
Project-scoped documentation and materials as agreed at kickoff.

Scope

Project Parameters and Customization

The following parameters are defined during project design. We work with you to match scope to your budget and timeline.

Parameter	Typical project scope
Host Cell Line	CHO-S, CHO-K1, HEK293, HEK293T, or custom mammalian suspension/adherent line
Transposon System	PiggyBac (classical or hyperactive) or Sleeping Beauty (SB100X); selected based on integration bias and efficiency
Vector Cargo Size	Scoped per project after consultation
Number of Clones Isolated	Scoped per project after consultation
Selection Marker	Puromycin, blasticidin, hygromycin, neomycin, or fluorescent marker (GFP, RFP)
Copy Number Control	Transposase-to-vector ratio titration for low, medium, or high copy number (as scoped)
Integration Site Analysis (Optional)	NGS-based mapping of transposon insertion sites (e.g., Tn-seq); available as add-on
Turnaround Time	Scoped per project after consultation

Workflow

How the Service Works

Our end-to-end process moves from design to deliverable in a structured, milestone-based manner.

Project Inception and Vector Design

We review your pathway genes, regulatory elements, and preferred host. Our bioinformatics team designs the optimal transposon vector layout, including ITR sequences, promoters, and selection markers.

Vector Construction and assessment

We synthesize and clone the complete transposon cassette into the backbone. The final plasmid is verified by restriction digest and full-insert Sanger sequencing across all junctions.

Transfection and Pool Generation

The vector and transposase are co-transfected (lipid-based or nucleofection) into the target cell line. After selection, we expand the surviving pool and assess initial expression levels. Pools are typically banked for long-term stability.

Single-Cell Cloning and Expansion

Cells are cloned via limiting dilution or FACS into 96-well plates. Clonal populations are expanded and screened for integration copy number and expression homogeneity. The best clones are advanced to cryopreservation.

QC Release and Final Report

We perform the agreed QC assays (qPCR, RT-qPCR, sterility, mycoplasma) on the final clone(s) or pool. A project report including methods, QC, and datasets is delivered along with cryovials and vector stocks.

Applications

Common Uses of Transposon-based Pathway Integration

This service supports a wide range of research and bioprocessing goals.

Stable Cell Lines for Recombinant Protein Production

Integrate metabolic pathways (e.g., glycolysis, glycosylation) or biosynthetic pathways for monoclonal antibodies, enzymes, and biologics. Transposon integration allows rapid generation of high-expressing clones without viral vectors.

Multi-gene metabolic engineering
Antibody heavy/light chain co-expression
Glycosylation enzyme pathways

Reporter and Functional Cell Lines

Engineer stable reporter cell lines with multi-component sensors (e.g., GPCR signaling cascades, fluorescent reporters) for high-throughput screening and drug discovery assays.

Transcription factor/promoter reporter constructs
Biosensor pathways (e.g., calcium/BRET)
Multiple reporters for multiplexed readouts

Model Cell Lines for Transposon-based Vectors

Generate stable cell lines expressing therapeutic genes or entire signaling pathways for in vitro testing of transposon vectors and gene therapy constructs.

Transposon vector validation in relevant cell types
Pathway complementation or knockout rescue models
Long-term expression studies

FAQ

Frequently Asked Questions

Common questions about our transposon integration service.

Both PiggyBac and Sleeping Beauty can carry inserts up to approximately 100 kb. For very large pathways, we recommend splitting into multiple transposons or using a single vector with internal promoters. The exact limit depends on the backbone and IP issues with some larger constructs; we will assess feasibility during the design phase.
Copy number is roughly controlled by adjusting the transposase-to-vector ratio. Higher transposase concentration tends to yield more multi-copy events, while lower ratios produce fewer integrations. Integration sites are semi-random (TTAA for PiggyBac, TA for Sleeping Beauty). If you require known integration sites or a narrow copy number range, we offer optional NGS-based mapping and can isolate clones with desired characteristics.
We provide a QC report that includes: integration copy number by qPCR, transgene expression by RT-qPCR (normalized to a housekeeping gene), Sanger sequencing of the ITR junctions, cell bank viability post-thaw, and sterility/mycoplasma testing. No guarantee of a specific expression level is made; the achievable titer depends on the pathway and regulatory elements used. Acceptance criteria are defined in the project SOW.
Yes, we routinely work with suspension-adapted CHO-K1, CHO-S, and HEK293 cells. If your host line is not suspension-adapted, we can adapt it prior to transfection (as an additional service). For cGMP-compatible cell banks, we follow internal quality protocols but cannot certify cGMP on our research service page—please discuss requirements with the project manager.

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