Earticle

초록

영어

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with α1,3-galactosyltransferase gene (α1,3-GT gene), DT-A/pGT5’/neo/pGT3’, DT- A/NLS/pGT5’/neo/pGT3’ and pGT5’/neo/ pGT3’/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5’ recombination arm (pGT5’) and a 1.9-kb fragment as the 3’ recombination arm (pGT3’). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of α1,3-GT locus. DT-A/pGT5’/neo/pGT3’ vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5’/neo/pGT3’ vector contain positive-negative selection marker and NLS sequences in upstream of 5’ recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5’/neo/pGT3’/NLS vector contain only positive selection marker and NLS sequence in downstream of 3’ recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with 300 μg/ml G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for α 1,3-GT gene disruption in 3´ PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5’/neo/pGT3’ knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

목차

ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
  Cloning of α1,3-GT Gene and Nuclear Localization Signal (NLS)
  Construction of α1,3-GT Knock-Out Vectors
  Preparation and Culture of Porcine Ear Fibroblast
  Transfection and Selection
  PCR Analysis of Targeted Cells
  Southern Blot Analysis of Targeted Cells
 RESULTS
 DISCUSSION
 REFERENCES

키워드

저자

• Hye Min Kim [ Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University, Gwangju 500-757, Korea, Alternative Toxicological Methods Research Center, Division of Predictive Toxicology, Korea Institute of Toxicology, Daejeon 350-343, Korea ]
• Sang Mi Lee [ Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University, Gwangju 500-757, Korea ]
• Hyo Young Park [ Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University, Gwangju 500-757, Korea, Laboratory of Reproductive Medicine, Creation and Love Women’s Hospital, Gwangju 502-800, Korea ]
• Man-Jong Kang [ Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University, Gwangju 500-757, Korea ] Corresponding author

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간행물

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• 간행물명

  Reproductive & developmental biology

• 간기

  계간

• pISSN

  1738-2432

• eISSN

  2288-0151

• 수록기간

  1977~2018

• 십진분류

  KDC 527 DDC 636