Abstract
In vivo delivery of morpholino antisense oligonucleotides (MAO) directly into the uterine lumen of a peri-implantation period pregnant sheep is an effective technique for evaluation of gene products for conceptus development. The highly phagocytic conceptus is undergoing rapid morphological change, thereby the available MAO are readily consumed and delivered to developing cells. Here, we describe the method for preparation and surgical delivery of MAO—Endo-Porter complex to developing ovine conceptus on day 8 postmating. Also outlined are methods for posttreatment sample recovery on day 16 postmating.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Guillomot M, Flechon JE, Wintenberger-Torres S (1981) Conceptus attachment in the ewe: an ultrastructural study. Placenta 2:169–182
Guillomot M (1995) Cellular interactions during implantation in domestic ruminants. J Reprod Fertil Suppl 49:39–51
Spencer TE, Johnson GA, Bazer FW, Burghardt RC (2007) Fetal-maternal interactions during the establishment of pregnancy in ruminants. Soc Reprod Fertil Suppl 64:379–396
Wu G, Bazer FW, Satterfield MC, Li X, Wang X, Johnson GA, Burghardt RC, Dai Z, Wang J, Wu Z (2013) Impacts of arginine nutrition on embryonic and fetal development in mammals. Amino Acids 45:241
Spencer TE, Johnson GA, Bazer FW, Burghardt RC (2004) Implantation mechanisms: insights from the sheep. Reproduction 128:657–668
Dunlap KA, Palmarini M, Varela M, Burghardt RC, Hayashi K, Farmer JL, Spencer TE (2006) Endogenous retroviruses regulate periimplantation growth and differentiation. Proc Natl Acad Sci 103:14390–14395
Wang W, Ying W, Dunlap KA, Lin G, Satterfield MC, Burghardt RC, Wu G, Bazer FW (2014) Arginine decarboxylase and agmatinase: an alternative pathway for de novo biosynthesis of polyamines for development to mammalian conceptuses. Biol Reprod 90:84
Wang W, Frank JW, Little DR, Dunlap KA, Satterfield MC, Burghardt RC, Hansen TR, Wu G, Bazer FW (2014) Functional role of arginine during the peri-implantation period of pregnancy I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm. FASEB J 28:2852–2863
Wang W, Frank JW, Wu J, Dunlap KA, Satterfield MC, Burghardt RC, Romero JJ, Hansen TR, Wu G, Bazer FW (2014) Functional role of arginine during the peri-implantation period of pregnancy II. Consequences of loss of function of nitric oxide synthase NOS3 mRNA in ovine conceptus trophectoderm. Biol Reprod 91:59
Luu KC, Nie GY, Salamonsen LA (2004) Endometrial calbindins are critical for embryo implantation: evidence from in vivo use of morpholino antisense oligonucleotides. Proc Natl Acad Sci 101:8028–8033
Ruiz-Gonzalez I, Minten M, Wang X, Dunlap KA, Bazer FW (2015) Involvement of TLR7 and TLR8 in conceptus development and establishment of pregnancy in sheep. Reproduction 149:305–316
Brooks K, Spencer TE (2015) Biological roles of interferon tau (IFNT) and type 1 IFN receptors in elongation of the ovine conceptus. Biol Reprod 92:1–10
Dorniak P, Bazer FW, Spencer TE (2011) Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium. Biol Reprod 84:119–1127
Schulte-Merker S, Stainer DYR (2014) Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology. Development 141:3103–3104
Brooks K, Burns G, Spencer TE (2015) Biological roles of hydroxysteroid (11-Beta) dehydrogenase 1(HSD11B1), HSD11B2, and glucocorticoid receptor (NR3C1) in sheep conceptus elongation. Biol Reprod 93:38
Brooks K, Burns G, Spencer TE (2015) Peroxisome proliferator activator receptor gamma (PPARG) regulates conceptus elongation in sheep. Biol Reprod 92:42
Acknowledgement
The authors would like to thank Fuller W. Bazer, M. Carey Satterfield, and Thomas E. Spencer, as well as former graduate students and postdoctoral fellows in the Bazer, Spencer and Satterfield laboratories for their participation in development of the in vivo MAO delivery method for use in pregnant sheep. This research was supported by the Agriculture and Food Research Initiative competitive grants 2011-6705-20028 and 2011–67015-20067 from the U.S. Department of Agriculture National Institute of Food and Agriculture, as well as support via National Institutes of Health grant HD052745.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Wang, X., Dunlap, K.A. (2017). Delivery of Morpholino Antisense Oligonucleotides to a Developing Ovine Conceptus via Luminal Injection into a Ligated Uterine Horn. In: Moulton, H., Moulton, J. (eds) Morpholino Oligomers. Methods in Molecular Biology, vol 1565. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6817-6_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6817-6_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6815-2
Online ISBN: 978-1-4939-6817-6
eBook Packages: Springer Protocols