Using light to measure gene expression in response to estrogen signaling

For you to understand the experiment I’m working on this week I need to provide some orientation to molecular genetics. Most everyone understands that genes are responsible for the characteristics we have. For example there are three possibilities for human hair texture; curly, wavy or straight. What is less well-know by non- or beginning biologists is that some genes are only turned on sometimes (biologists say expressed). For example we humans make a different type of hemoglobin (the protein that carries oxygen to individual cells) while developing in the uterus and getting oxygen indirectly from our mother’s blood than when we’re out here breathing air for ourselves. Thus there’s a mechanism that turns the gene for the in utero type off and the born-human type on near birth. Gene expression is regulated. Most everyone knows that genes are made of the chemical DNA and that the sequence of the famous letters A, T, G and C somehow leads to the characteristic like hair texture or type of hemoglobin (HB). The two types of HB are specified by different genes. The letter sequence of the DNA will be similar, after all both types of protein need to carry oxygen, but somewhat different. Here’s the important part of this story: DNA sequence is copied (transcribed) into mRNA and then the mRNA sequence is used as a pattern (translated) to make a sequence of amino acids and that’s the protein. Thus you can measure gene expression either by determining the amount or location of mRNA of a specific sequence or of a protein of a particular type. So in my immunofluorescence experiment I used the fact that the protein GFAP is expressed in astrocytes but not neurons to identify astrocytes. I had hoped to show that estrogen receptor alpha is expressed in neurons and not astrocytes but . . . One of the areas of biology that I find very interesting is what is called cell signaling. An example of a cell signal, that most everyone knows about are hormones. These molecules are released into the blood by glands and have effects on target cells. In order for a cell to be a target cell it must contain another molecule called a receptor; a protein that specifically sticks (biologists say binds) to that hormone. By the way, this specific binding stuff is a key molecular mechanism is biology. Antibodies bind to antigens; enzymes bind to substrates; signals bind to receptors; transcription factors bind to promoters (regions of DNA involved in beginning the production of specific RNAs). So cells that respond to the hormone signal estrogen contain estrogen receptors.One common mechanism of hormone action is to turn certain specific genes on (or off). So the expression of those specific genes is regulated by estrogen. Essentially it works like this: the estrogen receptors (ER) are present in the cytoplasm of estrogen-sensitive cells, estrogen (E₂ = estradiol, major chemical component of estrogen) diffuses from the blood into the cell and binds to two receptors so they form a dimer. The dimers move into the nucleus and bind to specific sections of the DNA called EREs (estrogen-response elements) turning the nearby genes on. More specifically this binding attracts (biologists say recruits) proteins (transcription factors) and the enzyme RNA polymerase so that mRNA is made (transcribed) from the specific gene. The mRNA moves out of the nucleus and is translated into a protein in the cytoplasm. Many proteins in many different cells are made under the direction of genes that are estrogen- sensitive. We could specifically look for those proteins in response to estrogen treatment. But we’re going to do something different to detect estrogen signaling by measuring the amount of light produced. More about the way that works next time. I’m going to use cultured cells to perform my study. That doesn’t mean that my cells are particularly musical or artistic. It means that the cells I’m going to study are growing in a dish; isolated from an animal. Many of us are interested in how things work in people or animals or even plants. Why would we want to study cells growing in a dish? The answer is that we can more easily control the amounts and types of molecules and other environmental influences (pH, temperature, number of cells) in a cell culture. The cells I’m using are Chinese hamster ovary (CHO) cells. Scientists have been working with these cells for many years. Originally someone isolated cells from the ovary of a Chinese hamster and figured out what mixture of water, ions, nutrients and so forth would allow the cells to grow in a dish. Now one buys the cells from a catalog and we know what medium to use (also purchased from a catalog). My mentor in Dr. Shannon Weickert’s lab is Dr. Jenny Wong. She’s an expert on the type of study I’m doing. In fact, I’m replicating something she’s done before. In this way I can learn the technique and know what the outcome “should” be. If I don’t get the same results either I’ve done something wrong or something else was not controlled as expected. Yesterday, I took a culture bottle on which CHO cells were happily growing and split them. I transferred some into another bottle, to keep the culture going and I transferred some into a multi-welled plate, a clear plastic dish with 48 little chambers and a top that covers all the chambers. For my experiment, I plated cells in 18 chambers. Friday I’m going to test the effects of six different concentrations of estrogen in triplicate. As anyone who’s done any kind of experiment or followed a recipe knows, there are a number of steps that you need to perform in a specific order. What is particularly challenging about this experiment is that everything must be kept completely sterile! We’re all cautioned to sneeze into a tissue and wash our hands. Bacteria and fungal spores are everywhere! We want to avoid them so we don’t get sick. I want to keep them out of my cultures because I want to measure the behavior of CHO cells and not CHO cells plus an unknown number of different types of bacteria and fungi. So we work in a special box (called a hood) that is sterilized with ultraviolet light before we start. The air flow is also controlled so that when the fan is on, air flows out, never in. We also spray the hood and any glass bottles we put in the hood with 70% alcohol. We wear gloves and even spray our gloves with 70% alcohol as well. All the plastic dishes and pipettes (special droppers that allow us to precisely measure the amount of liquid we use) are wrapped until we use them. We work with our arms inside the hood and we are careful never to touch, even with gloves, anything that will come into contact with our cells. I was glad to have Jenny there to stop me before I made an unconscious mistake. Learning the steps and getting your hands to do just exactly what they must do is very difficult. I understand intellectually what must be done but to actually do it effectively requires careful concentration and practice. It’s a little bit like that trick when you try to pat your nose and rub your stomach at the same time; only more so. I successfully platted my cells and observing them today showed no evidence of contamination. Today I changed the media. To change the medium, I had to draw the fluid out of each well, add and then draw off saline twice to get all the old medium without disturbing the cells and then add in new medium. My hands were shaking and I had to change pipettes because I made unapproved contacts. But, I don’t think I introduced any contamination. The culture medium we used to start the little cultures had fetal calf serum in it and we need to grow the cells for a day without that because it has estrogen in it and remember we want to control the amount of estrogen. Check back again. Transfection tomorrow, estrogen treatment on Thursday and looking for that revealing light on Friday.