Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Efficient Gene Targeting using Nuclear Localization Signal (NLS) and Negative Selection Marker Gene in Porcine Somatic Cells

  • Kim, Hye Min (Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University) ;
  • Lee, Sang Mi (Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University) ;
  • Park, Hyo Young (Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University) ;
  • Kang, Man-Jong (Department of Animal Science, College of Agriculture & Life Science, Insti. of Ag. Sci. and Tech., Chonnam National University)
  • Received : 2014.06.09
  • Accepted : 2014.06.18
  • Published : 2014.06.30
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Abstract

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 ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-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 ${\alpha}1,3$-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 $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-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.

Keywords

References

  1. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM (1998): Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells. Nat Biotechnol 16: 642-646. https://doi.org/10.1038/nbt0798-642
  2. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL (2002): Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 20:251-255. https://doi.org/10.1038/nbt0302-251
  3. Dean DA (1997): Import of plasmid DNA into the nucleus is sequence specific. Exp Cell Res 230:293-302. https://doi.org/10.1006/excr.1996.3427
  4. Dean DA, Dean BS, Muller S, Smith LC (1999): Sequence requirements for plasmid nuclear import. Exp Cell Res 253:713-722. https://doi.org/10.1006/excr.1999.4716
  5. Denning C, Priddle H (2003): New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells. Reproduction 126:1-11. https://doi.org/10.1530/rep.0.1260001
  6. Graessmann M, Menne J, Liebler M, Graeber I, Graessmann A (1989): Helper activity for gene expression, a novel function of the SV40 enhancer. Nucleic Acids Res 17:6603-6612. https://doi.org/10.1093/nar/17.16.6603
  7. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Da y BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002): Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089-1092. https://doi.org/10.1126/science.1068228
  8. Lundin C, Erixon K, Arnaudeau C, Schultz N, Jenssen D, Meuth M, Helleday T (2002): Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells. Mol Cell Biol 22:5869-5878. https://doi.org/10.1128/MCB.22.16.5869-5878.2002
  9. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo- derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348-352. https://doi.org/10.1038/336348a0
  10. Mir B, Piedrahita JA (2004): Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts. Nucleic Acids Res 32:e25. https://doi.org/10.1093/nar/gnh023
  11. Muller, U (1999): Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev 82:3-21. https://doi.org/10.1016/S0925-4773(99)00021-0
  12. Piedrahita JA, Moore K, Oetama B, Lee CK, Scales N, Ramsoondar J, Bazer FW, Ott T (1998): Generation of transgenic porcine chimeras using primordial germ cell-derived colonies. Biol Reprod 58:1321-1329. https://doi.org/10.1095/biolreprod58.5.1321
  13. Stice SL, Strelchenko NS, Keefer CL, Matthews L (1996): Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol Reprod 54:100-110. https://doi.org/10.1095/biolreprod54.1.100
  14. Takata M, Sasaki MS, Sonoda E, Morrison C, Hashimoto M, Utsumi H, Yamaguchi-Iwai Y, Shinohara A, Takeda S (1998): Homologous recombination and non-homologous end-joining pathways of DNA double- strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. Embo J 17:5497-5508. https://doi.org/10.1093/emboj/17.18.5497
  15. Templeton NS, Roberts DD, Safer B (1997): Efficient gene targeting in mouse embryonic stem cells. Gene Ther 4:700-9. https://doi.org/10.1038/sj.gt.3300457
  16. Wheeler MB (1994): Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev 6:563-568. https://doi.org/10.1071/RD9940563
  17. Yagi T, Nada S, Watanabe N, Tamemoto H, Kohmura N, Ikawa Y, Aizawa S (1993): A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Anal Biochem 214: 77-86. https://doi.org/10.1006/abio.1993.1459
  18. Yamaizumi M, Mekada E, Uchida T, Okada Y (1978): One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15:245-250. https://doi.org/10.1016/0092-8674(78)90099-5