Single-embryo Gene Expression for Early Embryo Development
Mylene Yao, M.D. Assistant Professor
Dept. of Obstetrics and Gynecology Stanford University
Mylene Yao, M.D., and fellow Stanford University researchers found that Oct4, the master regulator of embryonic stem cell pluripotency, also has critical functions during reprogramming of the early mammalian embryo. This research may ultimately lead to improvements in human in vitro fertilization methods and is relevant to stem cell and cancer research.
They recently published their findings in a research paper entitled, A Novel and Critical Role for Oct4 as a Regulator of the Maternal-Embryonic Transition, available at http://www.ncbi.nlm.nih.gov/pubmed/19129941.
“Scientists from around the world are looking at how to reprogram a highly differentiated somatic cell into a pluripotent embryonic stem cell-like cell,” Dr. Yao said. “There are many different approaches with up and down sides to each; such as, how do you do this without causing side effects like cancer and how can you apply it to therapeutics? The question we ask is very different. We think the mouse, or human embryo, is a good model to study reprogramming because this is the only situation in nature where you have highly specialized cell types—the egg and sperm—that fuse together to undergo reprogramming to produce a pluripotent cell, that is capable of differentiating into many cell types.”
The Stanford scientists focused their efforts on the reprogramming mechanisms used by the embryo that direct how pluripotency or totipotency is established. They picked the well-studied Oct4 gene for its known reprogramming functions and because it is highly expressed in the oocyte and early one- to two-cell embryo stage.
“We microinjected specific antisense morpholino oligonucleotides (morpholinos) to knock down the [Oct4] gene,” Dr. Yao said. “This is a well established approach in other model organisms but not so much in the mouse. So we had to establish the protocols and the controls and it turned out to be a powerful technique. We were able to get very specific gene knockdown and highly reproducible results.”
Their first finding was that Oct4 has a critical function prior to the blastocyst stage.
“That finding was new and we were very excited about it,”Dr. genes showing less inter-embryo variation are more tightly controlled… and we believe that these genes are very important in the gene network. What’s interesting is when we ranked them, the top two genes with the least amount of inter-embryo variation were Rest and Mta2. These genes were just reported to have important pluripotency functions in embryonic stem cells*. Mta2 is also implicated in human breast cancer. So it’s really exciting. Not only were we able to validate the gene chips results, but the BioMark helps us prioritize the genes for further study.”
The Research Experiment
To find out what the Oct4 pluripotency regulator does at the one- to two-cell stage, the researchers did gene chip experiments on the Affymetrix whole mouse genome chip and then Fluidigm’s Bio-Mark? System for Gene Expression Analysis.
The researchers ran a total of three independent experiments, each with an Oct4 knockdown sample and an uninjected sample. Each sample was derived from 20 embryos pooled together. Following RNA extraction using the PicoPure kit (Molecular Devices, they amplified half the pooled embryo sample with the Nugen Pico Ovation kit to prepare the samples for gene chip experiments. They repeated these steps for another three pairs of samples comprising cyclin A2 knockdown embryos and uninjected controls, as a positive control.
