Advertisement

Microfluidic-aided genotyping of zebrafish in the first 48 h with 100 % viability

Abstract

This paper introduces an innovative method for genotyping 1–2 days old zebrafish embryos, without sacrificing the life/health of the embryos. The method utilizes microfluidic technology to extract and collect a small amount of genetic material from the chorionic fluid or fin tissue of the embryo. Then, using conventional DNA extraction, PCR amplification, and high resolution melt analysis with fluorescent DNA detection techniques, the embryo is genotyped. The chorionic fluid approach was successful 78 % of the time while the fin clipping method was successful 100 % of the time. Chorionic fluid was shown to only contain DNA from the embryo and not from the mother. These results suggest a novel method to genotype zebrafish embryos that can facilitate high-throughput screening, while maintaining 100 % viability of the embryo.

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

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

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

References

  1. T. Bansal, J. Lenhart, T. Kim, C. Duan, M.M. Maharbiz, Biomed. Microdevices 11, 633–641 (2009). doi:10.1007/s10544-008-9273-5

  2. D. Choudhury, D. van Noort, C. Iliescu, B. Zheng, K.-L. Poon, S. Korzh, V. Korzh, H. Yu, Lab Chip 12(5), 892–900 (2012). doi:10.1039/c11c20351g

  3. D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Anal. Chem. 70, 4974–4984 (1998). doi:10.1021/ac980656z

  4. E. Fujimoto, T.J. Stevenson, C.B. Chien, J.L. Bonkowsky, Dev. Biol. 352, 393–404 (2011). doi:10.1016/j.ydbio.2011.01.023

  5. K. Howe et al., Nature 496, 498–503 (2013). doi:10.1038/nature12111

  6. D.-H. Kim, Y. Sun, S. Yun, B. Kim, C.N. Hwang, S.H. Lee, B. Nelson, Conf. Proc. IEEE Eng. Med. Biol. Soc. 7, 5061–5064 (2004)

  7. K.M. Kwan, E. Fujimoto, C. Grabher, B.D. Mangum, M.E. Hardy, D.S. Campbell, J.M. Parant, H.J. Yost, J.P. Kanki, C.-B. Chien, Dev. Dyn. 236, 3088–3099 (2007). doi:10.1002/dvdy.21343

  8. G. Lieschke, P.D. Currie, Nat. Rev. Genet. 8, 353–367 (2007). doi:10.1038/nrg2091

  9. M. Westerfield. 5th ed. University of Oregon Press, Eugene, (2007)

  10. J.M. Parant, S.A. George, R. Pryor, C.T. Wittwer, H.J. Yost, Dev. Dyn. 238, 3168–3174 (2009). doi:10.1002/dvdy.22143

  11. R. Samuel, C.M. Thacker, A.V. Maricq, B.K. Gale, J. Micromech. Microeng. 24, 105007 (2014). doi:10.1088/0960-1317/24/10/105007

  12. S.U. Son, R.L. Garrell, Lab Chip 9, 2398–2401 (2009). doi:10.1039/b906257b

  13. W. Wang, X. Liu, D. Gelinas, B. Ciruna, Y. Sun, PLoS One 2(9), e862 (2007). doi:10.1371/journal.pone.0000862

  14. E.M. Wielhouwer, S. Ali, A. Al-Afandi, M.T. Blom, M.B. Olde Riekerink, C. Poelma, J. Westerweel, J. Oonk, E.X. Vrouwe, W. Buesink, H.G.J. van Mil, J. Chicken, R. van’t Oever, M.K. Richardson, Lab Chip 11, 1815–1824 (2011). doi:10.1039/c01c00443j

  15. F. Yang, Z. Chen, J. Pan, X. Li, J. Feng, H. Yang, Biomicrofluidics 5, 024115 (2011). doi:10.1063/1.3605509

Download references

Acknowledgments

JLB and RS were supported by NIH DP2 MH100008. The authors would like to thank the Wittwer Lab, and the State of Utah Center of Excellence in Biomedical Microfluidics at the University of Utah, for providing technical resources and expertise.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

“All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.”

Author information

Correspondence to Joshua L. Bonkowsky or Bruce K. Gale.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(MOV 968 kb)

(MOV 813 kb)

ESM 1

(DOCX 2075 kb)

ESM 2

(MOV 968 kb)

ESM 3

(MOV 813 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Samuel, R., Stephenson, R., Roy, P. et al. Microfluidic-aided genotyping of zebrafish in the first 48 h with 100 % viability. Biomed Microdevices 17, 43 (2015). https://doi.org/10.1007/s10544-015-9946-9

Download citation

Keywords

  • Zebrafish
  • Genotyping
  • Microfluidics