Hostname: page-component-78c5997874-mlc7c Total loading time: 0 Render date: 2024-11-17T00:19:22.825Z Has data issue: false hasContentIssue false
  • English
  • Français

DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA–liposome complexes in IVF bovine zygotes

Published online by Cambridge University Press:  01 October 2012

G. Vichera ,
L.N. Moro ,
C. Buemo  and
D. Salamone
G. Vichera
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Capital Federal (C1417), Argentina.
L.N. Moro
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Capital Federal (C1417), Argentina.
C. Buemo
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Capital Federal (C1417), Argentina.
D. Salamone*
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Capital Federal (C1417), Argentina.
*
All correspondence to: D. Salamone. Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Capital Federal (C1417), Argentina. Tel: +54 0114524-8000. e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX–EGFP–liposome complexes or pBCKIP2.8–liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX–EGFP–liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP−) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin–liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.

Keywords

BovineDNA fragmentationin vitro fertilizationLiposomeTransgene expression
Type
Research Article
Information
Zygote , Volume 22 , Issue 2 , May 2014 , pp. 195 - 203
Copyright
Copyright © Cambridge University Press 2012 

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

Alikani, M., Calderon, G., Tomkin, G., Garrisi, J., Kokot, M. & Cohen, J. (2000). Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro. Hum. Reprod. 15, 2634–43.Google Scholar
Bavister, B. & Yanagimachi, R. (1977). The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod. 16, 228–37.Google Scholar
Betts, D.H. & King, W.A. (2001). Genetic regulation of embryo death and senescence. Theriogenology 55, 171–91.Google Scholar
Bondioli, K., Ramsoondar, J., Williams, B., Costa, C. & Fodor, W. (2001). Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Mol. Reprod. Dev. 60, 189–95.Google Scholar
Brackett, B. & Oliphant, G. (1975). Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12, 260–74.CrossRefGoogle ScholarPubMed
Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K. & Palmiter, R.D. (1985). Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82, 4438–42.CrossRefGoogle ScholarPubMed
Byrne, A.T., Southgate, J., Brison, D.R. & Leese, H.J. (1999). Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J. Reprod. Fertil. 117, 97–105.Google Scholar
Chan, A., Homan, J., Ballou, L., Burns, J. & Bremel, R. (1998). Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Agric. Sci. 95, 14028–33.Google ScholarPubMed
Chauhan, M., Nadir, S., Bailey, T., Pryor, A., Butler, S., Notter, D., Velander, W. & Gwazdauskas, F. (1999). Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct. J. Dairy Sci. 82, 918–26.Google Scholar
Cibelli, J., Stice, S., Gluekey, P., Kane, J., Jerry, J., Blackwell, C., Ponce de Leo¢n, A. & Robl, J. (1998). Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–8.Google Scholar
Cohen, G.M., Sun, X.M., Snowden, R.T., Dinsdale, D. & Skilleter, D.N. (1992). Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem. J. 286, 331–4.Google Scholar
Coleman, D.A., Dailey, R.A., Leffel, R.E. & Baker, R.D. (1986). Estrous synchronization and establishment of pregnancy in bovine embryo transfer recipients. J. Dairy Sci. 70, 858–66.Google Scholar
Collins, J.A., Schandi, C.A., Young, K.K., Vesely, J. & Willingham, M.C. (1997). Major DNA fragmentation is a late event in apoptosis. J. Histochem. Cytochem. 45, 923–34.Google Scholar
Eyestone, W.H. (1999). Production and breeding of transgenic cattle using in vitro embryo production technology. Theriogenology 51, 509–17.Google Scholar
Gandolfi, F., Terqui, M., Modina, S., Brevini, T. A., Ajmone-Marsan, P., Foulon-Gauze, F., & Courot, M. (1996). Failure to produce transgenic offspring by intra-tubal insemination of gilts with DNA-treated sperm. Reprod. Fertil. Dev. 8, 1055–60.Google Scholar
Golovan, S., Meidinger, R., Ajakaiye, A., Cottrill, M., Wiederkehr, M., Barney, D., Plante, C., Pollard, J., Fan, M., Hayes, M., Laursen, J., Hjorth, J., Hacker, R., Phillips, J. & Forsberg, C. (2001). Pigs expressing salivary phytase produce low-phosphorus manure. Nat. Biotechnol. 19, 741–5.Google Scholar
Hammer, R., Pursel, V., Rexroad, C. Jr, Wall, R., Bolt, D., Ebert, K., Palmiter, R. & Brinster, R. (1985). Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315, 20–6.CrossRefGoogle ScholarPubMed
Hardy, K.Cell death in the mammalian blastocyst. (1997). Mol. Hum. Reprod. 3, 919–25.Google Scholar
Holm, P., Booth, P., Schmidt, M., Greve, T. & Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum proteins. Theriogenology 52, 683–700.CrossRefGoogle ScholarPubMed
Ikawa, M., Kominamik, K., Yoshimura, Y., Tanaka, K., Nishimune, Y. & Okabe, M. (1995). A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP). FEBS Lett. 375, 125–8.CrossRefGoogle ScholarPubMed
Jaenisch, R. (1976). Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Cell Biol. 73, 1260–4.Google ScholarPubMed
Jurisicova, A., Varmuza, S. & Casper, R.F. (1996). Programmed cell death and human embryo fragmentation. Mol. Hum. Reprod. 2, 93–8.Google Scholar
Krimpenfort, P., Rademakers, A., Eyestone, W., Van derSchans, A., Van denBroek, S., Kooiman, P., Kootwijk, E., Platenburg, G., Pieper, F. & Strijker, R.et al. (1991). Generation of transgenic dairy cattle using ‘in vitro’ embryo production. Biotechnology (NY) 9, 844–7.Google Scholar
Lavitrano, M., Camaioni, A., Fazio, V., Dolci, S., Farace, M. & Spadafora, C. (1989). Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57, 717–23.CrossRefGoogle ScholarPubMed
Lindner, G.M. & Wright, R.W. Jr (1983). Bovine embryo morphology and evaluation. Theriogenology 20, 407–16.CrossRefGoogle ScholarPubMed
Mateusen, B., Van Soom, A., Maes, D.G., Donnay, I., Duchateau, L. & Lequarre, A.S. (2005). Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study. Reproduction 129, 443–52.CrossRefGoogle ScholarPubMed
Matwee, C., Betts, D.H. & King, W.A. (2000). Apoptosis in the early bovine embryo. Zygote 8, 57–68.Google Scholar
Murakami, M., Fahrudin, M., Varisanga, M.D. & Suzuki, T. (1999). Fluorescence expression by bovine embryos after pronuclear microinjection with the EGFP gene. J. Vet. Med. Sci. 61, 843–7.Google Scholar
Niemann, H., Lampeter, W.W., Sacher, B. & Kruff, B. (1982). Comparison of survival rates of day 7 and day 8 bovine embryos after fast freezing and thawing. Theriogenology 18, 445–52.Google Scholar
Pampfer, S., Vanderheyden, I., McCracken, J.E., Vesela, J. & De Hertogh, R. (1997). Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro. Development 124, 4827–36.Google Scholar
Paula-Lopes, F.F. & Hansen, P.J. (2002). Apoptosis is an adaptive response in bovine preimplantation embryos that facilitates survival after heat shock. Biochem. Biophys. Res. Commun. 295, 37–42.CrossRefGoogle ScholarPubMed
Perry, A., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyoda, Y. & Yanagimachi, R. (1999). Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, 1180–3.Google Scholar
Rideout, W.M. 3rd, Eggan, K. & Jaenisch, R. (2001). Nuclear cloning and epigenetic reprogramming of the genome. Science 293, 1093–8.Google Scholar
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., Fernandez, N., Papouchado, M., Foti, M., Judewicz, N., Mujica, I., Muñoz, L., Alvarez, S.F., Gonzalez, E., Zimmermann, J., Criscuolo, M. & 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.Google Scholar
SAS Institute (1989). ‘SAS/STAT: User's Guide. Version 6. Vol. 1. 4th edn.SAS Institute: Cary, NC, USA.Google Scholar
Schnieke, A., Kind, A., Ritchie, W., Mycock, K., Scott, A., Ritchie, M., Wilmut, I., Colman, A. & Campbell, K. (1997). Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278, 2130–3.Google Scholar
Stice, S., Robl, J., Ponce de Leon, F., Jerry, J., Golueke, P., Cibelli, J. & Kane, J. (1998). Cloning: new breakthrough leading to commercial opportunities. Theriogenology 49, 129–38.CrossRefGoogle ScholarPubMed
Szczygiel, M.A., Moisyadi, S. & Ward, W.S. (2003). Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol. Reprod. 68, 1903–10.Google Scholar
Tervit, H., Whittingham, D. & Rowson, L. (1972). Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 30, 493–7.Google Scholar
Vichera, G., Moro, L. & Salamone, D. (2010). Efficient transgene expression in ivf and parthenogenetic bovine embryos by intracytoplasmic injection of DNA–liposome complexes. Reprod. Dom. Anim. 46, 214–20.Google Scholar
Wall, R.J. (2001). Pronuclear microinjection. Cloning Stem Cells 3, 209–20.CrossRefGoogle ScholarPubMed
Wilmut, I., Archibald, A., Harris, S., McClenaghan, M., Simons, J., Whitelaw, C. & Clark, A. (1990). Modification of milk composition. J. Reprod. Fertil. Suppl. 41, 135–46Google Scholar
Yamauchi, Y., Doe, B., Ajduk, A. & Ward, M.A. (2007). Genomic DNA damage in mouse transgenesis. Biol. Reprod. 77, 803–12.Google Scholar