Utilizing the Labcyte Echo® Liquid Handler and Gateway® Gene Assembly in the Generation and Transfection of a PTEN-GFP Construct into a PTEN Null PC-3 Cell Line

The study of protein function relies upon recombinant gene assembly for protein expression and functional assays. This strategy is particularly important when available information regarding function is limited.  The vast amount of data generated from genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating vectors containing gene fusions, gene variants, genetic coding for protein tags and cellular targeting. Furthermore, efficient high throughput cloning and gene assembly procedures are essential for interrogating signaling pathways with multiple gene constructs. The resulting gene constructs can be analyzed in stable transgenic or in transiently expressing biological systems.

Utilizing the Labcyte Echo 525 liquid handler we demonstrate an efficient and robust gene assembly method to generate a fusion protein for functional cellular assays.  A PTEN-GFP construct was generated using the Gateway chemistry.  PTEN null PC-3 cells were transfected with this construct to generate a fluorescently labelled and functional PTEN cell line to interrogate the PI3K pathway. The assembled fusion protein now enables functional cellular assays including, but not limited to, viability and localization studies. The Echo 525 liquid handler enables flexibility and scalability for gene assembly while streamlining and automating the process.  The acoustic, non-contact dispense eliminates tips and potential for cross contamination. The low 25 nL resolution enables reduction in reaction volume which results in reduced reagent consumption and reduced costs for the user.