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Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases

  • Yuan, Yu-Guo (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Song, Shao-Zheng (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Zhu, Meng-Ming (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • He, Zheng-Yi (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Lu, Rui (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Zhang, Ting (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Mi, Fei (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Wang, Jin-Yu (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University) ;
  • Cheng, Yong (Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University)
  • Received : 2016.09.19
  • Accepted : 2016.12.13
  • Published : 2017.08.01

Abstract

Objective: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion: This finding signifies the combined use of TALENs and SCNT can generate biallelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

Keywords

Beta-lactoglobulin;Gene Targeted;Transcription Activator-Like Effector Nucleases;Human Lacotoferrin;Goat;Nuclear Transfer

References

  1. Meyer M, de Angelis MH, Wurst W, Kuh R. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci USA 2010;107:15022-6. https://doi.org/10.1073/pnas.1009424107
  2. McCreath KJ, Howcroft J, Campbell KH, et al. Production of genetargeted sheep by nuclear transfer from cultured somatic cells. Nature 2000;405:1066-9. https://doi.org/10.1038/35016604
  3. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows. Nat Commun 2013;4:2565. https://doi.org/10.1038/ncomms3565
  4. Liu X, Wang Y, Tian Y, et al. Generation of mastitis resistance in cows by targeting human lysozyme gene to ${\beta}$-casein locus using zinc-finger nucleases. Proc Biol Sci B 2014;281:20133368. https://doi.org/10.1098/rspb.2013.3368
  5. Zhang X, Wang L, Wu Y, et al. Knockout of myostatin by zinc-finger nuclease in sheep fibroblasts and embryos. Asian-Australas J Anim Sci 2016;29:1500-7. https://doi.org/10.5713/ajas.16.0130
  6. Yu B, Lu R, Yuan Y, et al. Efficient TALEN-mediated myostatin gene editing in goats. BMC Dev Biol 2016;16:26. https://doi.org/10.1186/s12861-016-0126-9
  7. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle. Transgenic Res 2015;24:147-53. https://doi.org/10.1007/s11248-014-9832-x
  8. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci USA 2012;109:17382-7. https://doi.org/10.1073/pnas.1211446109
  9. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous recombination in goats directs production of ${\beta}$-lactoglobulinfree, high-human lactoferrin milk. Sci Rep 2015;5:10482. https://doi.org/10.1038/srep10482
  10. Sharma S, Pravindra K, Betzel C, Singh TP. Structure and function of proteins involved in milk allergies. J Chromatogr B Biomed Sci Appl 2001;756:183-7. https://doi.org/10.1016/S0378-4347(01)00107-4
  11. Yu, S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Res 2011; 21:1638-40. https://doi.org/10.1038/cr.2011.153
  12. Jabed A, Wagner S, McCracken J, Wells DN, Laible G. Targeted microRNA expression in dairy cattle directs production of ${\beta}$-lactoglobulinfree, high-casein milk. Proc Natl Acad Sci USA 2012;109:16811-6. https://doi.org/10.1073/pnas.1210057109
  13. Yang B, Wang J, Tang B, et al. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle. PLoS One 2011;6:e17593. https://doi.org/10.1371/journal.pone.0017593
  14. Yu H, Chen J, Sun W, et al. The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct. J Biotechnol 2012;161:198-205. https://doi.org/10.1016/j.jbiotec.2012.06.035
  15. Yang P, Wang J, Gong G, et al. Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PLoS One 2008;3:e3453. https://doi.org/10.1371/journal.pone.0003453
  16. An LY, Yuan YG, Yu BL, Yang TJ, Cheng Y. Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure. Theriogenology 2012;78:1303-11. https://doi.org/10.1016/j.theriogenology.2012.05.027
  17. Cheng Y, An LY, Yuan GY, et al. Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice. Mol Reprod Dev 2012;79:573-85. https://doi.org/10.1002/mrd.22063
  18. Bertolini LR, Meade H, Lazzarotto CR, et al. The transgenic animal platform for biopharmaceutical production. Transgenic Res 2016;32:107-21.
  19. Guschin DY, Waite AJ, Katibah GE, et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 2010;649:247-56.
  20. Xu Y, Liu S, Yu G, et al. Excision of selectable genes from transgenic goat cells by a protein transducible TAT-Cre recombinase. Gene 2008; 419:70-4. https://doi.org/10.1016/j.gene.2008.04.020
  21. Yu H, Chen J, Liu S, et al. Large-scale production of functional human lysozyme in transgenic cloned goats. J Biotechnol 2013;168:676-83. https://doi.org/10.1016/j.jbiotec.2013.10.023
  22. Cho SW, Kim S, Kim Y, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 2014;24:132-41. https://doi.org/10.1101/gr.162339.113
  23. Lina L, Xu W, Dai Y, Li N. DNA methylation changes in cell line from ${\beta}$-lactoglobulin gene targeted fetus. Anim Reprod Sci 2009;112:402-8. https://doi.org/10.1016/j.anireprosci.2008.05.072
  24. Bui HT, Wakayama S, Kishigami S, et al. Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos. Bio Reprod 2010;83:454-63.
  25. Huan Y, Zhu J, Huang B, et al. Trichostatin A rescues the disrupted imprinting induced by somatic cell nuclear transfer in pigs. PLoS One 2015;10:e0126607. https://doi.org/10.1371/journal.pone.0126607
  26. Meng L, Wan Y, Sun Y, et al. Generation of five human lactoferrin transgenic cloned goats using fibroblast cells and their methylation status of putative differential methylation regions of IG$F_2$R and H19 imprinted genes. PLoS One 2013;8:e77798. https://doi.org/10.1371/journal.pone.0077798
  27. Tian XC. Genomic imprinting in farm animals. Annu Rev Anim Biosci 2014;2:23-40. https://doi.org/10.1146/annurev-animal-022513-114144
  28. Matoba S, Liu Y, Lu F, et al. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell 2014;159:884-95 https://doi.org/10.1016/j.cell.2014.09.055