Hostname: page-component-cd9895bd7-gxg78 Total loading time: 0 Render date: 2024-12-27T08:18:01.318Z Has data issue: false hasContentIssue false
  • English
  • Français

Disulphide-less crotamine is effective for formation of DNA–peptide complex but is unable to improve bovine embryo transfection

Published online by Cambridge University Press:  30 October 2019

Vicente J.F. Freitas Open the ORCID record for Vicente J.F. Freitas [Opens in a new window] ,
Iana S. Campelo ,
Mirelly M.A.S. Silva ,
Camila M. Cavalcanti ,
Dárcio I.A. Teixeira ,
Luiz S.A. Camargo ,
Luciana M. Melo  and
Gandhi Rádis-Baptista
Vicente J.F. Freitas*
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Iana S. Campelo
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Mirelly M.A.S. Silva
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Camila M. Cavalcanti
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Dárcio I.A. Teixeira
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Luiz S.A. Camargo
Affiliation:
Embrapa Dairy Cattle, Juiz de Fora, Brazil
Luciana M. Melo
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil Molecular Genetics Research Unit, University Center Fametro (UNIFAMETRO), Fortaleza, Brazil
Gandhi Rádis-Baptista*
Affiliation:
Laboratory of Biochemistry and Biotechnology, Institute of Marine Science, Federal University of Ceará (UFC), Fortaleza, Brazil
*
Authors for correspondence: Vicente J.F. Freitas. State University of Ceará, Av. Dr. Silas Mungusba, 1700 – Fortaleza, CE 60714-903, Brazil. Tel: +55 85 3101 9861. E-mail: vicente.freitas@uece.br; Gandhi Rádis-Baptista. Federal University of Ceará, Av. da Abolição, 3207 – Fortaleza – CE 60165-081, Brazil. Tel: +55 85 3366 7000, E-mail: radisbra@yahoo.com
Authors for correspondence: Vicente J.F. Freitas. State University of Ceará, Av. Dr. Silas Mungusba, 1700 – Fortaleza, CE 60714-903, Brazil. Tel: +55 85 3101 9861. E-mail: vicente.freitas@uece.br; Gandhi Rádis-Baptista. Federal University of Ceará, Av. da Abolição, 3207 – Fortaleza – CE 60165-081, Brazil. Tel: +55 85 3366 7000, E-mail: radisbra@yahoo.com
Get access Rights & Permissions [Opens in a new window]

Summary

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

Keywords

EmbryotoxicityGene deliveryMicroinjectionNucleic acid–peptide complexTransgenesis
Type
Research Article
Information
Zygote , Volume 28 , Issue 1 , February 2020 , pp. 72 - 79
Copyright
© Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Campelo, IS, Pereira, AF, Alcântara-Neto, AS, Canel, NG, Souza-Fabjan, JMG, Teixeira, DIA, Camargo, LS, Melo, LM, Rádis-Baptista, G, Salamone, DF and Freitas, VJF (2016a) Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos. Zygote 24, 48–57.CrossRefGoogle ScholarPubMed
Campelo, IS, Canel, NG, Bevacqua, RJ, Melo, LM, Rádis-Baptista, G, Freitas, VJF and Salamone, DF (2016b) Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression. J Assist Reprod Genet 33, 1405–13.CrossRefGoogle Scholar
Campos, VF, de Leon, PM, Komninou, ER, Dellagostin, OA, Deschamps, JC, Seixas, FK and Collares, T (2011) NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency. Theriogenology 76, 1552–60.CrossRefGoogle ScholarPubMed
Carballada, R, Relloso, M and Esponda, P (2002) Generation of transgenic mice by transfection of pronuclear embryos using lipid–DNA complexes. Zygote 10, 209–16.CrossRefGoogle ScholarPubMed
Chang, K, Qian, J, Jiang, M, Liu, YH, Wu, MC, Chen, CD, Lai, CK, Lo, HL, Hsiao, CT, Brown, L, Bolen, J Jr, Huang, HI, Ho, PY, Shih, PY, Yao, CW, Lin, WJ, Chen, CH, Wu, FY, Lin, YJ, Xu, J and Wang, K (2002) Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer. BMC Biotechnol 2, 5.CrossRefGoogle ScholarPubMed
Chen, PC, Hayashi, MAF, Oliveira, EB and Karpel, RL (2012) DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier. PLoS One 7, e48913.CrossRefGoogle Scholar
Ebert, KM, Selgrath, JP, DiTullio, P, Denman, J, Smith, TE, Memon, MA, Schindler, JE, Monastersky, GM, Vitale, JA and Gordon, K (1991) Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression. Biotechnology 9, 835–8.Google Scholar
Fadel, V, Bettendorff, P, Herrmann, T, de Azevedo, WF , Jr, Oliveira, EB, Yamane, T and Wüthrich, K (2005) Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus . Toxicon 46, 759–767.CrossRefGoogle ScholarPubMed
Freitas, VJF, Alcântara-Neto, AS, Pereira, AF, Campelo, IS, Melo, LM and Rádis-Baptista, G (2014) Assessing the complex formation between crotamine – a natural cell penetrating peptide – and DNA using high sensitive fluorescence exclusion assay. Clon Transgen 3, 128.Google Scholar
Garrels, W, Ivics, Z and Kues, WA (2012) Precision genetic engineering in large mammals. Trends Biotechnol 30, 386–93CrossRefGoogle ScholarPubMed
Gonçalves, JM and Polson, A (1947) The electrophoretic analysis of snake venoms. Arch Biochem 13, 253–9.Google ScholarPubMed
Gordon, JW, Scangos, GA, Plotkin, DJ, Barbosa, JA and Ruddle, FH (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77, 7380–4.CrossRefGoogle ScholarPubMed
Hacker, DL and Balasubramanian, S (2016) Recombinant protein production from stable mammalian cell lines and pools. Curr Opin Struct Biol 38, 129–36.CrossRefGoogle ScholarPubMed
Hammer, RE and Pursel, VG (1985) Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315, 680–3.CrossRefGoogle ScholarPubMed
Iqbal, K, Barg-Kues, B, Brol, S, Bode, J, Niemann, H and Kues, WA (2009) Cytoplasmic injection of circular plasmids allows targeted expression in mammalian embryos. Biotechniques 47, 959–68.CrossRefGoogle ScholarPubMed
Jaenisch, R and Mintz, B (1974) Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl Acad Sci USA 71, 1250–4.CrossRefGoogle ScholarPubMed
Kerkis, A, Kerkis, I, Rádis-Baptista, G, Oliveira, EB, Vianna-Morgante, AM, Pereira, LV and Yamane, T (2004) Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus . FASEB J 18, 1407–9.CrossRefGoogle ScholarPubMed
Kim, JS (2016) Genome editing comes of age. Nat Protoc 11, 1573–8.CrossRefGoogle ScholarPubMed
Krimpenfort, P, Rademakers, A, Eyestone, W, van der Schans, A, van den Broek, S, Kooiman, P, Kootwijk, E, Platenburg, G, Pieper, F, Strijker, R and de Boer, H (1991) Generation of transgenic dairy cattle using in vitro embryo production. Biotechnology 9, 844–7.Google Scholar
Kues, WA and Niemann, H (2011) Advances in farm animal transgenesis. Prev Vet Med 102, 146–56.CrossRefGoogle ScholarPubMed
Li, R, Miao, J, Fan, Z, Song, S, Kong, IK, Wang, Y and Wang, Z (2018) Production of genetically engineered golden Syrian hamsters by pronuclear injection of the CRISPR/Cas9 complex. J Vis Exp 131, 56263.Google Scholar
Malaweera, DBO, Ramachandra, S, Wu, JB, Oh, SK, Kim, SH, Kim, SJ, Jang, G and Cho, JK (2014) Establishment of efficient microinjection system in the porcine embryos. J Emb Transf 29, 59–66.CrossRefGoogle Scholar
Marinovic, MP, Dal Mas, C, Monte, GG, Felix, D, Campeiro, JD and Hayashi, MAF (2016) Crotamine: function diversity and potential applications. In Gopalakrishnakone, P, Inagaki, H, Mukherjee, A, Rahmy, T and Vogel, CW (eds) Snake venoms. Dordrecht: Springer.Google Scholar
Meng, F, Li, H, Wang, X, Qin, G, Oback, B and Shi, D (2015) Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection. J Anim Sci Biotechnol 6, 44.CrossRefGoogle ScholarPubMed
Nascimento, FD, Hayashi, MA, Kerkis, A, Oliveira, V, Oliveira, EB, Rádis-Baptista, G, Nader, HB, Yamane, T, Tersariol, IL and Kerkis, I (2007) Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem 282, 21349–60.CrossRefGoogle ScholarPubMed
Nicastro, G, Franzoni, L, de Chiara, C, Mancin, AC, Giglio, JR and Spisni, A (2003) Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom. Eur J Biochem 270, 1969–79.CrossRefGoogle ScholarPubMed
Pooga, M and Langel, Ü (2015) Classes of cell-penetrating peptides. Methods Mol Biol 1324, 3–28.CrossRefGoogle ScholarPubMed
Rádis-Baptista, G and Kerkis, I (2011) Crotamine, a small basic polypeptide myotoxin from rattlesnake venom with cell-penetrating properties. Curr Pharm Des 17, 4351–61.CrossRefGoogle ScholarPubMed
Rádis-Baptista, G, de la Torre, BG and Andreu, D (2008) A novel cell-penetrating peptide sequence derived by structural minimization of a snake toxin exhibits preferential nucleolar localization. J Med Chem 51, 7041–4.CrossRefGoogle ScholarPubMed
Rádis-Baptista, G, de la Torre, BG and Andreu, D (2012) Insights into the uptake mechanism of NrTP, a cell-penetrating peptide preferentially targeting the nucleolus of tumour cells. Chem Biol Drug Des 79, 907–15.CrossRefGoogle ScholarPubMed
Rádis-Baptista, G, Campelo, IS, Morlighem, JRL, Melo, LM and Freitas, VJF (2017) Cell-penetrating peptides (CPPs): from delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis. J Biotechnol 252, 15–26.CrossRefGoogle ScholarPubMed
Rieth, A, Pothier, F and Sirard, MA (2000) Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events. Mol Reprod Dev 57, 338–45.3.0.CO;2-K>CrossRefGoogle ScholarPubMed
Salamone, D, Barañao, L, Santos, C, Bussmann, L, Artuso, J, Werning, C, Prync, A, Carbonetto, C, Dabsys, S, Munar, C, Salaberry, R, Berra, G, Berra, I, Fernández, N, Papouchado, M, Foti, M, Judewicz, N, Mujica, I, Muñoz, L, Alvarez, SF, González, E, Zimmermann, J, Criscuolo, M and Melo, C (2006) High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow. J Biotechnol 124, 469–72.CrossRefGoogle ScholarPubMed
Stewart, KM, Horton, KL and Kelley, SO (2008) Cell-penetrating peptides as delivery vehicles for biology and medicine. Org Biomol Chem, 6, 2242–55.CrossRefGoogle ScholarPubMed
Vichera, G, Moro, L and Salamone, D (2011) Efficient transgene expression in IVF and parthenogenetic bovine embryos by intracytoplasmic injection of DNA–liposome complexes. Reprod Domest Anim 46, 214–20.CrossRefGoogle ScholarPubMed
Xu, YN, Uhm, SJ, Koo, BC, Kwon, MS, Roh, JY, Yang, JS, Choi, HY, Heo, YT, Cui, XS, Yoon, JH, Ko, DH, Kim, T and Kim, NH (2013) Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes. J Genet Genomics 40, 37–43.CrossRefGoogle ScholarPubMed

Freitas et al. supplementary material

Freitas et al. supplementary material

Download Freitas et al. supplementary material(Video)
Video 4.4 MB