Multigene expression in stable CHO cell pools generated with the piggyBac transposon system

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Engineering

Background:

• Traditional gene delivery methods for recombinant protein production can be inefficient.
• Generating cell lines expressing multiple proteins often faces challenges in stability and yield.
• The piggyBac (PB) transposon system offers a potential solution for stable transgene integration.

Purpose of the Study:

• To evaluate the efficiency of the piggyBac (PB) transposon system for generating heterogeneous cell pools expressing multiple transgenes.
• To compare protein yields from PB-derived cell pools with those from conventional gene delivery methods.
• To investigate the impact of using single versus multiple selection agents on transgene integration and protein production.

Main Methods:

• Generation of recombinant Chinese hamster ovary (CHO) cell pools using the piggyBac (PB) transposon system.
• Stable expression of 1-4 transgenes encoding model proteins: enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody.
• Utilizing separate PB donor plasmids with identical or distinct selection genes for transgene delivery.
• Analysis of recombinant protein yields, transgene copy numbers, and integration patterns.

Main Results:

• PB-derived cell pools demonstrated higher recombinant protein yields compared to conventionally generated cell pools.
• When using a single selection agent, increased transgene numbers led to decreased protein production and integrated copies per transgene.
• The total number of integrated transgenes remained consistent regardless of the number of different transgenes when using a single selection agent.
• Employing distinct selection agents for each transgene resulted in an increased total number of integrated transgenes with a higher number of transfected transgenes.

Conclusions:

• The generation of cell pools expressing multiple recombinant proteins using the piggyBac (PB) transposon system is feasible.
• This method offers improved efficiency for recombinant protein production compared to conventional techniques.
• Optimizing transgene integration and expression for multi-protein production is most effective when each transgene is selected with a unique marker.