ABSTRACT
Gene transfer into isolated populations of cells provides valuable insight and is often a rst step in the investigation of a novel protein. Studies in cell lines often require gene transfer of a designated cDNA to evaluate the structure and/or function of a speci c protein without the compensatory adaptations often observed in intact animal models.1 Gene transfer at the cellular level also has proven enormously important toward achieving this goal under a variety of circumstances. One important
32.1 Introduction .......................................................................................................................... 557 32.1.1 Overview ................................................................................................................... 557 32.1.2 Gene Transfer by Transfection.................................................................................. 558 32.1.3 Vector-Mediated Gene Transfer ............................................................................... 558 32.1.4 Optimal Gene Transfer ............................................................................................. 558
32.2 Non-viral-Mediated Gene Transfer ...................................................................................... 559 32.2.1 Transfection Approaches for Gene Transfer ............................................................. 559 32.2.2 Newer Alternatives ...................................................................................................560 32.2.3 Calcium Phosphate Transfection Protocol ................................................................ 561
32.2.3.1 Reagents and Buffers ................................................................................. 561 32.2.3.2 Maintenance and Preparation of Mammalian Cells .................................. 561 32.2.3.3 Preparation of DNA ................................................................................... 562 32.2.3.4 Calcium Phosphate Transfection ............................................................... 562
32.3 Viral-Vector-Based Gene Transfer ........................................................................................ 563 32.3.1 Ideal Traits for Viral Vectors Used for Gene Transfer ............................................. 563 32.3.2 Currently Available Vectors ..................................................................................... 563
32.3.2.1 Retrovirus/Lentivirus................................................................................. 563 32.3.2.2 Adenovirus ................................................................................................. 565 32.3.2.3 AAVs .......................................................................................................... 567 32.3.2.4 Protocol for the Preparation of Recombinant Adenovirus ........................ 567 32.3.2.5 Introduction/Overview ............................................................................... 567 32.3.2.6 Generation of Plasmids for Recombination ............................................... 567 32.3.2.7 Cell Maintenance and Splitting Procedures of HEK293 Cells ................. 568
32.3.3 Production of Recombinant Adenoviruses: Homologous Recombination ............... 568 32.3.3.1 Noble Agar Overlay ................................................................................... 569 32.3.3.2 Expansion of Plaque-Puri ed Recombinant Adenovirus .......................... 570
32.4 Alternative Techniques ......................................................................................................... 576 References ...................................................................................................................................... 576
example is the use of gene transfer into genetically de ned animal models to gain insights into a speci c signaling pathway and/or function of a pathway during a speci c phase of organ development.2 In addition, currently available immortalized cardiac cells do not adequately model terminally differentiated adult cells.3 Thus, gene transfer into primary cultures of these cells provides a rst step toward characterizing and evaluating protein function. While the use of genetically modi ed animals, including transgenic and gene-targeted models, is a common practice, cellular gene transfer can often be utilized as a screen to determine the most appropriate model to pursue in intact animals.4,5 Gene transfer into cell lines or primary culture is a highly ef cient and effective approach for introducing a foreign piece of DNA into cells as a means to establish proof of concept or testing hypothesis-driven experiments.5
