title: Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts.
abstract: OBJECTIVE
  Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.
  
  
  METHODS
  In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 µg DNA per pulse in the first experiment and increments of 10 µg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR)-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.
  
  
  RESULTS
  The results showed no significant difference among three first concentrations except for group 1 (10 µg/pulse) and group 3 (30 µg/pulse), but there was a significant difference between these groups and the fourth group (p<0.05), as this group (40 µg/pulse) statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH) results indicated the transgene was integrated in a single position in PCR positive cells.
  
  
  CONCLUSIONS
  Increasing amount of using the vector 40µg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency.
authors.affiliation: 1. Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
authors.forename: Amir
authors.initials: A
authors.lastname: Amiri Yekta
authors.forename: Azam
authors.initials: A
authors.lastname: Dalman
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Sanati
authors.forename: Nayeralsadat
authors.initials: N
authors.lastname: Fatemi
authors.forename: Hamed
authors.initials: H
authors.lastname: Vazirinasab
authors.forename: Alireza
authors.initials: A
authors.lastname: Zomorodipour
authors.forename: Mohammad
authors.initials: M
authors.lastname: Chehrazi
authors.forename: Hamid
authors.initials: H
authors.lastname: Gourabi
keywords: Electroporation; Fibroblast; Gene Transfer; Naked DNA; Transgenic Animals