title: Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII.
abstract: OBJECTIVE
  Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.
  
  
  METHODS
  This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.
  
  
  RESULTS
  The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.
  
  
  CONCLUSIONS
  The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.
authors.affiliation: Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil. Electronic address: marcelafreitas@usp.br.
authors.forename: Marcela Cristina
authors.initials: MC
authors.lastname: Correa de Freitas
authors.affiliation: Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors.forename: Aparecida Maria
authors.initials: AM
authors.lastname: Fontes
authors.affiliation: Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors.forename: Andrielle
authors.initials: A
authors.lastname: de Castilho Fernandes
authors.affiliation: Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors.forename: Virginia
authors.initials: V
authors.lastname: Picanço-Castro
authors.affiliation: Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors.forename: Elisa Maria
authors.initials: EM
authors.lastname: de Sousa Russo
authors.affiliation: Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors.forename: Dimas Tadeu
authors.initials: DT
authors.lastname: Covas
keywords: Factor VIII; Hemophilia A; Virus integration