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College Park, MD, United States

Chen C.H.,National Taiwan University | Chen C.H.,Animal Technology Institute Taiwan | Du F.,Nanjing Normal University | Du F.,Renova Life, Inc. | And 13 more authors.
Theriogenology | Year: 2013

The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits. © 2013 Elsevier Inc.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 155.99K | Year: 2013

DESCRIPTION (provided by applicant): Project Summary We propose to develop a non-invasive method to predict mammalian oocyte quality by analyzing the surrounding cumulus cells' gene expression patterns (cGEP). The result of the cGEP assay---a cGEP score---can be used to predict embryo quality and ultimately the embryo transfer (ET) results. This technology, if success, will for the first time, provide a quantitative measurement of the in vitro fertilized IVF) embryos. It will help users to non-invasively identify the best embryo for transfer. Single embryo transfer is expected to decrease the frequencies of multiple gestations (i.e., increase the frequencies of singletons) in human IVF practices. Multiple gestations are associated with low birth weight babies, preterm births and maternal complications. While the increasing use of Assisted Reproductive Technology (ART) over the past two decades has helped thousands of infertile couples to have children, almost one third of ART pregnancies are twins or higherorder multiple gestations; 51% of all ART neonates are the products of multiple gestations, a frequency 15- to 20-fold greater than with spontaneous conceptions. In many IVF and intracytoplasmic sperm injection (ICSI) procedures, more than one embryo is transferred to ensure satisfactory pregnancy results. This is largely due to the inability to accurately select good embryos by morphology examinations alone, the major judgment method in most IVF clinics. In fact, up to 40% of embryos with normal morphology have abnormal chromosomes. Such practices (transferring multiple embryos) contribute greatly to the high percentage of multiple gestations in ART. The ultimate goal of the present project is to develop a non-invasive method to predict oocyte and embryo quality in human IVF practices, in a 24-48 h turnover time. With this technology, when oocytes are collected and subjected to IVF procedures, the cumulus cells from each individual oocyte can be used for cumulus gene expression pattern (cGEP) assay. A cGEP score will be generated for each oocyte; consequently, each embryo generated from this IVF session will have a corresponding cGEP score. The cGEP score will assist users to make wise decisions on how and when to transfer the embryos. To our knowledge, this application represents one of the first commercial attempts to develop a non-invasive technology to predict human embryo quality. Cumulus cells are abundant and are considered to be disposable byproducts of oocyte retrieval for IVF treatment. Collection and analysis of cumulus cells is non-invasive and does not harm the oocyte. Biomarkers from cumulus cells can be used for diagnosis of infertility or reproductive toxicity. They can be used to improve the efficiency and efficacy of infertility treatmentsby identifying the most viable oocytes for fertilization in vitro, as well as exclusion of bad embryos for transfer. This will reduce the ned for and side effects associated with hyperstimulation of ovarian development during IVF. By limiting the numberof oocytes needed for fertilization, fewer extra embryos will be generated, which avoids the ethical and social complications of handling and disposing of these extra embryos. It will also reduce the number of embryos transferred, thus reducing the risk associated with high-order multiple gestations. Multiple gestations are associated with low birth weight babies, preterm births and maternal complications 1. Reducing the frequencies of multiple gestations would not only reduce these clinical complicationsof the mothers and the newborns, but also reduce the medical costs associated with them. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: We propose to develop a non-invasive method to predict mammalian oocyte quality by analyzing the surrounding cumulus cells' gene expression patterns (cGEP). This technology will help users to non-invasively identify the best embryo for transfer. Single embryo transfer is expected to decrease the frequencies of multiple gestations (i.e., increase the frequenciesof singletons) in human IVF practices.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.13M | Year: 2010

DESCRIPTION (provided by applicant): The rabbit is a good animal model for the study of many human diseases because of its anatomical, genetic and biochemical similarities to the human. It is also used for drug screening, production of antibody and production of therapeutic proteins. The rabbit is large enough to provide adequate quantities of tissue for experimental work, produce sufficient amount of antibody or therapeutic proteins, and yet it is small enough to be economical for most laboratories. To date, pronuclear microinjection remains the primary method to produce transgenic rabbits. There are no germline competent rabbit embryonic stem (rbES) cell lines available. Pronuclear microinjection, however, is of low efficiency, results in random integration, and cannot produce gene targeted transgenic animals. The purpose of this SBIR project is to establish germline competent rbES cell lines for the production of gene targeted transgenic rabbits. In Phase I we established rabbit embryonic fibroblasts as feeder layers, synthesized recombinant rabbit leukemia inhibitory (LIF) factor, and derived putative rbES cells. In Phase II, we will work to validate if these rbES cells can contribute to germ line cells after blastocyst injection and we will work to produce gene targeted transgenic rabbits from these cells. Specific Aims in Phase II project include: (1) derive new rbES cell lines with inhibitors of differentiation; (2) evaluate germline competency of rbES cells; and (3) produce gene targeted transgenic rabbits. The success of this project will establish the first germline competent ES cell lines in a non-murine species, improve the transgenic efficiency in rabbits, and make the production of gene targeted rabbits possible. PUBLIC HEALTH RELEVANCE: The rabbit is a good animal model for the study of many human diseases because of its anatomical, genetic and biochemical similarities to the human. It is also used for drug screening, production of antibody and production of therapeutic proteins. The main reason that rabbit has not been in these fields is that there have been no established germline competent rabbit embryonic stem (rbES) cell lines. The success of this project will establish the first germline competent ES cell lines in a non-murine species, improve the transgenic efficiency in rabbits, and make the production of gene targeted rabbits possible.


Renova Life, Inc. | Entity website

Transgenic Rabbit Service Species: Rabbit Models: CVD, AIDS, Diabetes Service: Production and breeding of transgenic rabbits Timeline: Six months to two years. >>More Gene Editing in Rabbit and Guinea Pig Species: Rabbit and guinea pig Service:genome editing knockout and knockin Timeline: Four months to eight months ...


Renova Life, Inc. | Entity website

Transgenic Rabbit Service Species: Rabbit Models: CVD, AIDS, Diabetes Service: Production and breeding of transgenic rabbits Timeline: Six months to two years. >>More Gene Editing in Rabbit and Guinea Pig Species: Rabbit and guinea pig Service:genome editing knockout and knockin Timeline: Four months to eight months ...

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