title: A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro.
abstract: We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific gene osterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/ Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency, transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an increase in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.
authors.affiliation: Musculoskeletal Disease Center, JL Pettis Memorial Veterans Administration Medical Center, Loma Linda University, Loma Linda, CA 92357, USA.
authors.forename: Weirong
authors.initials: W
authors.lastname: Xing
authors.forename: David
authors.initials: D
authors.lastname: Baylink
authors.forename: Anil
authors.initials: A
authors.lastname: Kapoor
authors.forename: Subburaman
authors.initials: S
authors.lastname: Mohan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology*; D014162:Transfection / Q000379:methods*; D014780:Viruses / Q000235:genetics