Skip to main content

Advertisement

SpringerLink
Log in

Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 106 cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1–24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Li XC, Yue H, Li CY, He XH, Zhao QJ, Ma YH, Guan WJ, Ma JZ (2009) Establishment and characterization of a fibroblast cell line derived from Jining Black Grey goat for genetic conservation. Small Ruminant Res 87:17–26

    Article  Google Scholar 

  2. Na RS, Zhao QJ, da Jin P, Su XH, Chen XW, Guan WJ, Ma YH (2010) Establishment and biological characteristics of Ujumqin sheep fibroblast line. Cytotechnology 62(1):43–52

    Article  PubMed  CAS  Google Scholar 

  3. Qi Y, Tu Y, Yang D, Chen Q, Xiao J, Chen Y, Fu J, Xiao X, Zhou Z (2007) Cyclin A but not cyclin D1 is essential for c-myc-modulated cell-cycle progression. J Cell Physiol 210(1):63–71

    Article  PubMed  CAS  Google Scholar 

  4. Gu Y, Li H, Miki J, Kim KH, Furusato B, Sesterhenn IA, Chu WS, McLeod DG, Srivastava S, Ewing CM, Isaacs WB, Rhim JS (2006) Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines. Exp Cell Res 312(6):831–843

    Article  PubMed  CAS  Google Scholar 

  5. Suemori H, Yasuchika K, Hasegawa K, Fujioka T, Tsuneyoshi N, Nakatsuji N (2006) Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. Biochem Biophys Res Commun 345(3):926–932

    Article  PubMed  CAS  Google Scholar 

  6. Kawarai S, Hashizaki K, Kitao S, Nagano S, Madarame H, Neo S, Ishikawa T, Furuichi M, Hisasue M, Tsuchiya R, Tsujimoto H, Yamada T (2006) Establishment and characterization of primary canine hepatocellular carcinoma cell lines producing alpha-fetoprotein. Vet Immunol Immunopathol 113(1–2):30–36

    Article  PubMed  CAS  Google Scholar 

  7. Guan WJ, Ma YH, Zhou XY, Liu GL, Liu XD (2005) The establishment of fibroblast cell line and its biological characteristic research in Taihang black goat. Rev China Agric Sci Technol. 7:25–33

    Google Scholar 

  8. Hu PF, Guan WJ, Li XC, Ma YH (2012) Construction of recombinant proteins for reprogramming of endangered Luxi cattle fibroblast cells. Mol Biol Rep 39(6):7175–7182

    Article  PubMed  CAS  Google Scholar 

  9. Wu H, Guan W, Li H, Ma Y (2008) Establishment and characteristics of white ear lobe chicken embryo fibroblast line and expression of six fluorescent proteins in the cells. Cell Biol Int 32(12):1478–1485

    Article  PubMed  CAS  Google Scholar 

  10. Liu C, Guo Y, Guan W, Ma Y, Zhang HH, Tang X (2008) Establishment and biological characteristics of Luxi cattle fibroblast bank. Tissue Cell 40(6):417–424

    Article  PubMed  CAS  Google Scholar 

  11. Stacey GN (1997) Standardization in animal cell technology. Folia Microbiol (Praha) 42(2):113–116

    Article  CAS  Google Scholar 

  12. Ansari HA, Bosma AA, Broad TE, Bunch TD, Long SE, Maher DW, Pearce PD, Popescu CP (1999) Standard G-, Q-, and R-banded ideograms of the domestic sheep (Ovis aries): homology with cattle (Bos taurus). Report of the committee for the standardization of the sheep karyotype. Cytogenet Cell Genet 87(1–2):134–142

    Article  PubMed  CAS  Google Scholar 

  13. Frankel AD, Pabo CO (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55(6):1189–1193

    Article  PubMed  CAS  Google Scholar 

  14. Mäe M, Langel U (2006) Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery. Curr Opin Pharmacol 6(5):509–514

    Article  PubMed  Google Scholar 

  15. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Schöler HR, Duan L, Ding S (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4(5):381–384

    Article  PubMed  CAS  Google Scholar 

  16. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4(6):472–476

    Article  PubMed  CAS  Google Scholar 

  17. Brooks H, Lebleu B, Vivès E (2005) Tat peptide-mediated cellular delivery: back to basics. Adv Drug Deliv Rev 57(4):559–577

    Article  PubMed  CAS  Google Scholar 

  18. Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12(4):400–406

    Article  PubMed  CAS  Google Scholar 

  19. Han K, Jeon MJ, Kim KA, Park J, Choi SY (2000) Efficient intracellular delivery of GFP by homeodomains of Drosophila Fushi-tarazu and Engrailed proteins. Mol Cells 10(6):728–732

    PubMed  CAS  Google Scholar 

  20. Yoon HY, Lee SH, Cho SW, Lee JE, Yoon CS, Park J, Kim TU, Choi SY (2002) TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells. Neurochem Int 41(1):37–42

    Article  PubMed  CAS  Google Scholar 

  21. Kwon HY, Eum WS, Jang HW, Kang JH, Ryu J, Ryong Lee B, Jin LH, Park J, Choi SY (2000) Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells. FEBS Lett 485(2–3):163–167

    Article  PubMed  CAS  Google Scholar 

  22. Jin LH, Bahn JH, Eum WS, Kwon HY, Jang SH, Han KH, Kang TC, Won MH, Kang JH, Cho SW, Park J, Choi SY (2001) Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells. Free Radic Biol Med 31(11):1509–1519

    Article  PubMed  CAS  Google Scholar 

  23. Han K, Jeon MJ, Kim SH, Ki D, Bahn JH, Lee KS, Park J, Choi SY (2001) Efficient intracellular delivery of an exogenous protein GFP with genetically fused basic oligopeptides. Mol Cells 12(2):267–271

    PubMed  CAS  Google Scholar 

  24. Eum WS, Choung IS, Li MZ, Kang JH, Kim DW, Park J, Kwon HY, Choi SY (2004) HIV-1 Tat-mediated protein transduction of Cu, Zn-superoxide dismutase into pancreatic beta cells in vitro and in vivo. Free Radic Biol Med 37(3):339–349

    Article  PubMed  CAS  Google Scholar 

  25. Zorko M, Langel U (2005) Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv Drug Deliv Rev 57(4):529–545

    Article  PubMed  CAS  Google Scholar 

  26. Kang WH, Simon MJ, Gao S, Banta S, Morrison B III (2011) Attenuation of astrocyte activation by TAT-mediated delivery of a peptide JNK inhibitor. J Neurotrauma 28(7):1219–1228

    Article  PubMed  Google Scholar 

  27. Lee YJ, Johnson G, Pellois JP (2010) Modeling of the endosomolytic activity of HA2-TAT peptides with red blood cells and ghosts. Biochemistry 49(36):7854–7866

    Article  PubMed  CAS  Google Scholar 

  28. Schwarze SR, Hruska KA, Dowdy SF (2000) Protein transduction: unrestricted delivery into all cells? Trends Cell Biol 10(7):290–295

    Article  PubMed  CAS  Google Scholar 

  29. Nagel F, Dohm CP, Bähr M, Wouters FS, Dietz GP (2008) Quantitative evaluation of chaperone activity and neuroprotection by different preparations of a cell-penetrating Hsp70. J Neurosci Methods 171(2):226–232

    Article  PubMed  CAS  Google Scholar 

  30. Mukhopadhyay A (1997) Inclusion bodies and purification of proteins in biologically active forms. Adv Biochem Eng Biotechnol 56:61–109

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was funded by the Ministry of Agriculture of China for Transgenic Research Program (2011ZX08009-003-006, 2011ZX08012-002-06), the project National Infrastructure of Animal Germplasm Resources (2012 year), China Agriculture Research System-40-01 and Scientific Research Foundation for Doctors, Liaoning Province of China (20111148).

Author information

Authors and Affiliations

  1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

    P. F. Hu, W. J. Guan, X. C. Li, W. X. Zhang, C. L. Li & Y. H. Ma

  2. Institute of Wind Sandy Land Improvement and Utilization, Liaoning Academy of Agricultural Sciences, Fuxin, 123000, China

    P. F. Hu

Authors
  1. P. F. Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. J. Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. X. C. Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. W. X. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. L. Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Y. H. Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Y. H. Ma.

Additional information

P. F. Hu and W. J. Guan contributed equally to this study.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, P.F., Guan, W.J., Li, X.C. et al. Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat. Mol Biol Rep 40, 327–336 (2013). https://doi.org/10.1007/s11033-012-2064-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-012-2064-3

Keywords

Search

Navigation