PMID- 23577306
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20130412
LR  - 20211021
IS  - 2228-5806 (Print)
IS  - 2228-5814 (Electronic)
IS  - 2228-5806 (Linking)
VI  - 14
IP  - 4
DP  - 2013 Winter
TI  - Optimization of The Electroporation Conditions for Transfection of Human Factor 
      IX into The Goat Fetal Fibroblasts.
PG  - 270-5
AB  - 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. MATERIALS AND 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 microg DNA per pulse in the first experiment and 
      increments of 10 microg/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 microg/pulse) and group 3 (30 microg/pulse), but there was a significant 
      difference between these groups and the fourth group (p<0.05), as this group (40 
      microg/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. CONCLUSION: Increasing 
      amount of using the vector 40microg/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.
FAU - Amiri Yekta, Amir
AU  - Amiri Yekta A
AD  - 1. Department of Genetics at Reproductive Biomedicine Research Center, Royan 
      Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
FAU - Dalman, Azam
AU  - Dalman A
FAU - Sanati, Mohammad Hossein
AU  - Sanati MH
FAU - Fatemi, Nayeralsadat
AU  - Fatemi N
FAU - Vazirinasab, Hamed
AU  - Vazirinasab H
FAU - Zomorodipour, Alireza
AU  - Zomorodipour A
FAU - Chehrazi, Mohammad
AU  - Chehrazi M
FAU - Gourabi, Hamid
AU  - Gourabi H
LA  - eng
PT  - Journal Article
DEP - 20130220
PL  - Iran
TA  - Cell J
JT  - Cell journal
JID - 101566618
PMC - PMC3593931
OTO - NOTNLM
OT  - Electroporation
OT  - Fibroblast
OT  - Gene Transfer
OT  - Naked DNA
OT  - Transgenic Animals
EDAT- 2013/04/12 06:00
MHDA- 2013/04/12 06:01
PMCR- 2013/01/01
CRDT- 2013/04/12 06:00
PHST- 2012/01/04 00:00 [received]
PHST- 2012/07/16 00:00 [accepted]
PHST- 2013/04/12 06:00 [entrez]
PHST- 2013/04/12 06:00 [pubmed]
PHST- 2013/04/12 06:01 [medline]
PHST- 2013/01/01 00:00 [pmc-release]
PST - ppublish
SO  - Cell J. 2013 Winter;14(4):270-5. Epub 2013 Feb 20.