Synthesis and characterization of biologically active recombinant elk and horse FSH.
Abstract: The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well.
Publication Date: 2009-05-18 PubMed ID: 19500922DOI: 10.1016/j.anireprosci.2009.05.007Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Animal Models
- Animal Studies
- Assisted Reproductive Techniques
- Biotechnology
- Cell Culture
- Cloning
- Equine Health
- Equine Medicine
- Equine model
- Equine Research
- Equine Science
- Equine Studies
- Experimental Methods
- In Vitro Research
- Laboratory Methods
- Molecular biology
- Reproductive Technology
- RNA
- Veterinary Research
- Veterinary Science
Summary
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This research investigation focuses on the cloning and expression of the alpha-subunit and FSH beta-subunit cDNAs from elk and horse, and the production of recombinant FSH from these species in vitro. The study also explores the impact of these recombinant products on ovarian growth when injected into immature female Wistar rats.
Methodology
- The researchers extracted RNAs from the pituitary glands of elk and horse.
- These RNAs were then reverse-transcribed and amplified by a technique known as a polymerase chain reaction.
- The cDNAs or complementary DNAs corresponding to both subunits of FSH (Follicle Stimulating Hormone) in elk and horse were cloned.
- These cDNAs were then inserted into the expression vector pBudCE4.1 and transfected into CRL-9096 cells, a type of cell line.
Analysis of Gene Expression
- The expression of these genes was determined in the transfected cells through Northern and Western blot analysis, techniques used for detection of specific molecules in a sample.
Characterization of Recombinant FSH
- The recombinant FSH secreted in the culture media from these cells was then characterized using an in vitro bioassay and RIA (radioimmunoassay), a method used to measure concentrations of antibodies.
- The study found that the recombinant horse FSH was 5.6 times more potent than the elk FSH when assessed by their activity over mass.
Impact on Ovarian Growth
- These recombinant products were injected into immature female Wistar rats to observe the effect on ovarian growth.
- The injections stimulated ovarian growth in these rats, suggesting these products are, indeed, recombinant versions of the native elk and horse FSH.
Significance of the Study
- This study indicates that these recombinant products may be used to improve methods of ovarian stimulation in cervids, equids, and other species, contributing to more predictable and efficient techniques in the field of reproductive biology.
Cite This Article
APA
Fachal MV, Furlan M, Clark R, Card CE, Chedrese PJ.
(2009).
Synthesis and characterization of biologically active recombinant elk and horse FSH.
Anim Reprod Sci, 117(3-4), 331-340.
https://doi.org/10.1016/j.anireprosci.2009.05.007 Publication
Researcher Affiliations
- Department of Biology, University of Saskatchewan College of Art and Sciences, Saskatoon, Canada.
MeSH Terms
- Animals
- CHO Cells
- Cells, Cultured
- Cloning, Molecular
- Cricetinae
- Cricetulus
- DNA, Complementary / isolation & purification
- Follicle Stimulating Hormone / analysis
- Follicle Stimulating Hormone / biosynthesis
- Follicle Stimulating Hormone / genetics
- Follicle Stimulating Hormone / physiology
- Horses / genetics
- Recombinant Proteins / analysis
- Recombinant Proteins / biosynthesis
- Recombinant Proteins / genetics
- Ruminants / genetics
- Transfection
Citations
This article has been cited 2 times.- Elyasi Gorji Z, Amiri-Yekta A, Gourabi H, Hassani S, Fatemi N, Zerehdaran S, Vakhshiteh F, Sanati MH. Cloning and Expression of Iranian Turkmen-thoroughbred Horse Follicle Stimulating Hormone in Pichia pastoris. Iran J Biotechnol 2015 Jun;13(2):10-17.
- Gifre L, Arís A, Bach À, Garcia-Fruitós E. Trends in recombinant protein use in animal production. Microb Cell Fact 2017 Mar 4;16(1):40.
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