Evaluation of the Serratia marcescens nuclease (NucA) as a transgenic cell ablation system in porcine.
The efficiency of the Serratia marcescens nuclease encoded by the NucA gene, with or without a nuclear localization signal (NLS), and the commonly used diphtheria toxin A (DTA) were compared for their ability to ablate cells in culture. Constructs containing the test genes driven by the beta-actin promoter coupled with enhancer elements from the cytomegalovirus promoter and rabbit beta-globin gene (pCAG) and the blasticidin resistance gene driven by the phosphoglycerate kinase (PGK) promoter were generated and electroporated into porcine fetal fibroblasts. Three independent replicates were completed. Following blasticidin selection, the number of surviving colonies was counted to assess the efficiency of the toxic gene. Both NucA and DTA proved to be effective in killing porcine fibroblasts compared to controls. However, the efficiency of cell ablation was significantly higher with DTA than with NucA or NucANLS (p < 0.05). Gene expression analysis of surviving colonies indicated that survival is related to low or absent expression of the toxic genes. These results indicate that the NucA gene, while capable of mammalian cell ablation, is less efficient than DTA.
Caballero
I
I
Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, USA.
Piedrahita
J A
JA
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D011817:Rabbits; D012705:Serratia / Q000201:enzymology*; D013552:Swine
D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease