“We picked 42 genes to validate on the BioMark system,” Dr. Yao said. “We picked them to represent different functional categories.”
“We used the Fluidigm 48.48 dynamic array to validate differential gene expression between knockdown and control samples,” Dr. Yao explained.
Of the 42 genes, three were removed from the study due to technical difficulties, but expression changes of 39 of the genes were measured. Of those, 87% showed altered expression due to the Oct 4 knockdown. “We were very happy to be able to validate our results,” Dr. Yao said. “That level of scrutiny is very important for a strategy that is not widely used in the mouse model.”
Prior to using the BioMark highthroughput system, which can test as little material as a single cell against 96 genes, the group used conventional RT-PCR practices.
"Our single-embryo data allowed us to go beyond simply validating our gene chip data,” the researchers wrote in their paper. “Methods using samples comprised of pooled cells or embryos, generate relative gene expression that represents an average of all cells assayed, but they cannot discern between genes that are consistently differentially regulated versus those with a tendency towards stochastic changes; similarly, rare outlier embryos expressing unique transcriptomes are not recognized. By analyzing quantitative expression data at the single-embryo level, we were able to make this discrimination.”
Dr. Yao said prior to using the BioMark system, they only tested three genes from pooled samples. “In the conventional system, we would have to pool, say 10 embryos per sample to test two to three genes, and for genes not highly expressed, we’d have to pool 20. Semi qPCR is able to do it, but to see differential expression levels that are statistically significant, you’d have to repeat the experiment many more times or you’d have to pool many more samples.” Dr. Yao said the time savings in using the BioMark system was significant.
"What we accomplished on the BioMark system in three weeks would take a similarly skilled and dedicated researcher more than nine months to accomplish,” she said.
Possible Medical Application of Results
Dr. Yao said studying embryo development in mice, may ultimately help improve in vitro fertilization methods for treating human infertility.
"In clinical infertility, we don’t understand why some couples have trouble conceiving,” she said. “According to the diagnostic tests we have available, about 10-30% don’t have a cause that can be identifi ed.
Many embryos that are cultured do not make it to
the eight-cell stage by day three, or the blastocyst stage. We don’t
know if it is something inherent in thecouples or if the problem is that
in vitro fertilization methods are not really suited to culturing some human embryos.”
Dr.
Yao explained that it is technically not feasible to do gene chip
analysis on human embryos and it is good to first understand the mouse
system to see if we can understand what genes are critical for the
embryo development process, and to use that knowledge to investigate
clinical infertility.
Further Embryo Research Planned
Dr. Yao and her fellow researchers are now looking at the mouse embryo development at the multicell stage, by which time ~90% of Oct4 knockdown embryos have arrested in development.
“We’re going to do gene chip experiments at the other stages and use the BioMark to validate results and further deconstruct the gene network.”
Biography
Mylene W. M. Yao is assistant professor and clinician-scientist in obstetrics and gynecology, reproductive endocrinology and infertility, and reproductive biology. Dr. Yao graduated from medical school at University of Toronto in 1993, and completed her obstetrics and gynecology residency training at McGill University in 1998. She received her clinical subspecialty training in reproductive endocrinology and infertility at Brigham and Women’s Hospital, Harvard University, from 1998-2001. During that time, she also trained in developmental biology as post-doctoral fellow in the laboratory of Richard L. Maas, M.D., Ph.D. After a brief time as assistant professor at Columbia University, she moved to Stanford University in 2003 to initiate her independent research on mammalian embryo development.
Research Summary
We are interested to understand how the early mammalian embryo is reprogrammed to establish totipotent or pluripotent blastomeres, which subsequently undergo lineage-specific differentiation to give rise to all the cell types in the organism. Specifically, we aim to understand how key processes such as nuclear reprogramming, establishment of developmental competence, maintenance of pluripotency, and cell fate decisions are regulated at the earliest stages of mouse and human development. These questions are especially intriguing and relevant to medicine because the zygote, or one-cell stage embryo, are formed by the fusion of the sperm and egg, which are arguably the most differentiated cell types, as they only host haploid genomes. This natural process of reprogramming is relatively efficient, quick and safe from oncogenic potential. Thus, the early pre-blastocyst stages hold the key to Nature’s reprogramming toolkit, which is invaluable to our efforts in developing stem cell-based therapies. We have taken innovative approaches to establish both in vitro and in vivo human and mouse models, to identify the genetic and physical determinants that are critical and sufficient for optimal development. Our methods include gene knockdown strategy combined with single-embryo gene expression profi ling, applying microfluidics to control physical culture parameters, and the use of clinical in vitro fertilization (IVF) outcomes analysis to direct us towards clinically relevant embryo and reproductive phenotypes. We discovered novel and critical roles of master pluripotency regulator, Oct4, in the maternal-embryonic transition, and have begun to deconstruct the gene regulatory network in the early embryo. Combined with deep phenotyping efforts in early human embryo development and clinical IVF, we are investigating early human development in a translational context that is directly relevant to human health and disease. Our mission is to contribute new and essential knowledge to advance paradigms in the fields of stem cell, cancer, and assisted reproductive technologies, and broaden our scope of human development, health and disease in the 21st century.
原文下载：http://www.fluidigm.com/pdf/fldm/FLDM_MRKT00117.pdf
References and Related Links
Foygel, K., Choi, B., Jun, S., et al., 2008 Dec
31. A Novel and Critical Role for Oct4 as a Regulator of the
Maternal-Embryonic Transition. PLoS ONE.3(12): e4109.
http://www.ncbi.nlm.nih.gov/pubmed/19129941
Singh SK, Kagalwala MN,
Parker-Thornburg J, Adams H, Majumder S (2008) REST maintains
self-renewal and pluripotency of embryonic stem cells. Nature 453:
223-227. www.time.com; “Predicting In Vitro Success” by Alice Park; July
1, 2008: http://www.time.com/time/health/article/0,8599,1819524,00.html
National
Public Radio, “Predicting In Vitro Success Made Easier” by Joe Palca in
All Things Considered; aired July 3, 2008:
http://www.npr.org/templates/story/story.php?storyId=92204512
Reuters;
New method may help predict IVF success: study by Julie Steenhuysen:
http://www.reuters.com/article/scienceNews/idUSN0125903720080702
The
Guardian; “Fertility: Doctors find test to predict chances of IVF
success” by Ian Sample; July 2, 2008:
http://www.guardian.co.uk/science/2008/jul/02/medicalresearch.health
U.S.
News & World Report; “New Method Better Predictor of In Vitro
Fertilization Success”; July 1, 2008:
http://health.usnews.com/articles/health/healthday/2008/07/01/new-method-better-predictor-of-in-vitro.html
Dr. Yao’s home page http://womenshealth.stanford.edu/research/yao.html
