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Taking Short Cuts: How Direct Reprogramming Can Transform One Type of Cell Straight into Another

Difficulty
Time Required Short (2-5 days)
Prerequisites Basic understanding of how genes are turned into proteins.
Material Availability Readily available
Cost Very Low (under $20)
Safety No issues

Abstract

In the first decade of the 21st century, scientists found ways to make one adult cell type turn into a completely different cell type. This has huge implications for the medical field, including being able to take some cells that a person could spare, such as skin cells or blood cells, and turn them into another cell type that might be much more important for that person to have, such as cells to make a new kidney. How are scientists able to accomplish this amazing feat of "reprogramming" the identity of human cells? What potential problems will have to be solved before this technology can be used regularly in hospitals? In this science project, you will explore how scientists use small proteins called transcription factors to turn one cell type into another and what hurdles must be overcome to make this powerful practice more commonplace.

Objective

Determine how transcription factors can be used to directly change one cell type into another cell type.

Credits

Teisha Rowland, PhD, Science Buddies

  • Learn.Genetics is a registered trademark of the University of Utah Genetic Science Learning Center.

Cite This Page

MLA Style

Science Buddies Staff. "Taking Short Cuts: How Direct Reprogramming Can Transform One Type of Cell Straight into Another" Science Buddies. Science Buddies, 9 Oct. 2014. Web. 24 Oct. 2014 <http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioMed_p009.shtml>

APA Style

Science Buddies Staff. (2014, October 9). Taking Short Cuts: How Direct Reprogramming Can Transform One Type of Cell Straight into Another. Retrieved October 24, 2014 from http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioMed_p009.shtml

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Last edit date: 2014-10-09

Introduction

In the first decade of the 2000s, scientists found that they could turn one type of adult cell into multiple, completely different cell types. They do this by controlling which genes the cell uses. Genes are made up of many building blocks of DNA (deoxyribonucleic acid), our genetic code. Every cell in your body has the same genes, but the reason that your eyes are different from your kidneys, or that you do not grow hair on your palms, is that not all cell types use the same genes. This means that different cell types express different genes.

When a cell expresses a gene, it turns that gene into protein. The first major part of this process is called transcription, which is when the DNA for a gene is turned into mRNA (messenger ribonucleic acid). The mRNA is then turned into protein in the second major part of this process, called translation.

Small proteins called transcription factors regulate the transcription process. Transcription factors bind directly to DNA and, based on where they bind, this tells the cell which genes should be expressed. Transcription factors even regulate the expression of other transcription factors. Because different transcription factors are active in different cell types, different cell types make different proteins, and consequently have different identities.

A major breakthrough for using transcription factors to "reprogram" cell identity came in 2007, when two different research groups found that they could use four different transcription factors to turn mature, adult human cells into cells that were like human embryonic stem cells (hESCs). Human embryonic stem cells are isolated from early-stage embryos, called blastocysts, that were fertilized in laboratory conditions only about four or five days earlier (at this time in a normal pregnancy, the embryo would not yet be implanted in the uterus). At this point, the fertilized egg contains only about 150 cells and is a hollow, fluid-filled sphere. Because these cells are isolated at such an early stage during development, they can multiply and become any cell type found in the human body, whether bone, hair, heart, etc. Cells such as hESCs that can give rise to any cell type in the body are referred to as pluripotent (from the Latin words plurimus, meaning "very many," and potens, meaning "having power"). The reprogrammed cells are labeled induced pluripotent stem cells (iPSCs). To make iPSCs, the researchers mentioned above forced normal, adult cells to make just four transcription factors that are essential for the identity of hESCs. The transcription factors reprogrammed the identity of the adult cells. This was a significant breakthrough, because the ability of hESCs to turn into virtually any adult cell type gives them immense medical potential. Other types of stem cells, such as adult stem cells, are much more limited in what they can become.

Not only could the newly developed iPSCs turn into any adult cell type, like hESCs, but these new cells could also be patient specific. This means that a patient could have some of his or her cells removed, reprogrammed into iPSCs by forcing these cells to make hESC-related transcription factors, and then have the iPSCs turned into whatever types of cells the patient needs, such as cells for a new kidney. Making patient-specific cells like this may avoid some potential complications that can occur with tissue and organ transplants, such as immune rejection. A patient's immune system rejects a transplanted tissue or organ if the immune system recognizes the tissue or organ as being foreign. This is a normal, and usually healthy, process, since the body has to fight off foreign invaders all the time, such as viral and bacterial infections. But this normal process can cause serious problems because it can be hard to "convince" the body that a transplant is not a threat.

Although iPSCs have immense potential, they are still a relatively new technology and have some barriers that must be overcome before they can be used medically. However, their development helped renew interest in using transcription factors to give a cell a different identity. For example, in 2010, scientists reported that they could use three different transcription factors to turn mouse tail cells directly into neurons. Similar findings have been reported using other groups of transcription factors. This process of direct reprogramming, or turning one cell type directly into another type of cell, skips the need to have the iPSC middle man. This means that, theoretically, a patient could have some cells, such as skin cells, removed; these cells could then be directly made into the cell type the patient needs, such as liver cells for a liver transplant. All of this research is part of the rapidly developing field of regenerative medicine, which develops technologies that may be used to regenerate tissues and whole organs.

How do scientists pick which transcription factors to use? The scientists select a target cell type - the type of cell that they want to create. They then look at what transcription factors these cells normally make. At the same time, they select a cell type that they can start out with, a tissue that a person can easily donate, such as blood. Once they have done this research, they can take the starting cell type and force it to make the transcription factors that their target cell type normally makes. Ideally, these transcription factors will transform the cell type into their target cell type. Of course, this is a simplification of the process. In practice, many complications can make it difficult to directly reprogram cells successfully.

In this science project, you will be using online bioinformatics databases to explore how researchers select transcription factors to directly reprogram one cell type into another, desired (or target) cell type.

Terms and Concepts

  • Genes
  • DNA
  • Gene expression
  • Protein
  • Transcription
  • Translation
  • Transcription factors
  • Human embryonic stem cells (hESCs)
  • Blastocyst
  • Pluripotent
  • Induced pluripotent stem cells (iPSCs)
  • Stem cells
  • Patient-specific cells
  • Immune rejection
  • Direct reprogramming
  • Regenerative medicine
  • Bioinformatics
  • Microarray

Questions

  • How does a gene become expressed? What regulates its expression?
  • What are transcription factors?
  • How are transcription factors used to make induced pluripotent stem cells (iPSCs)?
  • Why may iPSCs be better for use in transplants than human embryonic stem cells (hESCs)?
  • Why may direct reprogramming be better than making iPSCs?
  • Other than blood, what cell types do you think would be good to use as "starting material" for direct reprogramming, cell types that the human body could most easily regenerate or spare?
  • What cell types do you think would be good target cell types, ones that are in high demand for transplants?

Bibliography

To do this science project you will need to use these databases:

These resources are good places to start gathering information about gene transcription, stem cells, transcription factors, and direct reprogramming:

The 2010 study on directly reprogrammed mouse tail cells into neurons:

Information about the transcription factors in Table 1 was taken from these sources:

  • Hu, Z. and S. Gallo. (2010, January 19). Identification of interacting transcription factors regulating tissue gene expression in human. Retrieved September 14, 2011, from http://www.biomedcentral.com/1471-2164/11/49
  • Yu, X., et al. (2006, September 18). Computational analysis of tissue-specific combinatorial gene regulation: Predicting interaction between transcription factors in human tissues. Retrieved September 14, 2011, from http://nar.oxfordjournals.org/content/34/17/4925.full

Materials and Equipment

  • Computer with an Internet connection
  • Lab notebook

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Experimental Procedure

Finding Transcription Factors in Your Target Cell Type

What types of cells are in the highest demand for tissue transplants? In other words, what types of cells are good targets for reprogramming efforts? For this science project, you can use any cell type shown in Table 1 as your target cell type. To simplify the directions, we will use kidney cells as the target cell type example throughout this Experimental Procedure. Kidneys are one of the most commonly transplanted organs, although many other types of tissues and organs are in high demand for transplants. In this part of the science project, you will investigate which transcription factors are made by your target cell type and are the most important for its identity.

  1. Select a tissue type listed in Table 1. This table was generated from published scientific studies that investigated which transcription factors are made and are active in different tissue types. Transcription factors usually have long names that are abbreviated for simplicity's sake, and those abbreviations are listed for each tissue type in Table 1.
    1. For data on organs donated for transplants to give you an idea of which tissues are in high demand, search this national data report from the Organ Procurement and Transplantation Network and the U.S. Department of Health and Human Services.
Tissue Type Important Transcription Factors
BloodELF1, PEA3, ETF, NRF1, FOXJ2, CEBP, PPARA, SP3, ETS, YY1, NFAT, PLZF
Bone marrow ETF, NRF1, MAX, SREBP1, STAT3, ELK1, CEBPA, DR4, SPZ1, OCT4, VDR, SREBP, DR3, WT1
Brain* ETF, CEBP, PBX1, LBP1, CHX10, FREAC7, GATA, OCT4, PPARA, POU3F2, NFAT
Colon AFP1, HNF1, CDP, FOXO4, CDP
Heart AP1, MEF2, RSRFC4, FOXJ2, POU3F2, SRF, MYOGNF1, SP3, CP2, ZIC3, TAXCREB, MAF, FAC1, OCT1
Kidney HNF1, HNF4, CRX, GCNF, HNF3, PPARA, SP3, ER, TEL2, SREBP
Liver CDP, HNF1, CEBP, E4BP4, HNF4, AIRE, HNF3, HNF3B, RUSH1A
Lung ETF, MYC, MAX, LBP1, TBP, NRF1, LHX3, EBOX, ETS, CEBP, SP3, WT1, CACD, CETS1P54, SPZ1
Lymph node ELK1, PU.1, ICSBP, NRF1, LBP1, LRF, OCT4, PAX4, CP2, ZIC3, TTF1, PPARA
Breast E2F, SRY, ETF, TAX, CREB, ZID, FAC1, RSRFC4
Muscle AP-4, MEF2, MYOD, RSRFC4, LBP1, OCT1, SP1, MYOGNF1, DR4, NKX25, TBP, TEL2, ZIC3, CP2, OCT, PAX2, TST1, CEBP, POU3F2, TST1, EGR1
Ovary VDR, CREB, MAZ, DBP, MZF1, ETF, CEBPA, SREBP1, GATA_C, MAF, LRF, NKX25, NF1, ZIC3, CHOP, PAX4
Pancreas ATF, HEB, NF-muE1, NRL, MYOD, E12, HNF3, TBP, DEC, MYOGNF1, GATA4, PAX4, YY1
Placenta ATF1, CHX10, LHX3, CEBPA, SP3, WT1, DEC, PAX5, NKX25, STAT1, PPARA, SP1
Prostate AFP1, LHX3, CART1, TBP, SRF
Skin AREB6, ALX4, ARP-1, E47, AREB6, VDR, LMO2, CEBPA, PPARA, HMGIY, OCT4, PLZF, CP2
Small intestine CART1, LHX3, HNF1, NKX6-2
Spleen E12, GCM, NFKB1, LBP1, MYOD, LBP1, RSRFC4
Spinal cord ETS, HMGIY, HIC1, PPARA, NKX25, PLZF, TBP, CART1, MYOGNF1
Stomach ETF, AREB6, NFE2, ER, HIFI, BACH1
Testicle AP2, NRF1, EGR1, EGR3, NF-Y, SP1, DR4, SPZ1, POU3F2, PAX4, MYOGNF1, ZIC3, MYOGNF1
Thymus ATF, TAX, CREB, ETF, NRF1, EBOX, CEBPA, AP1, STAT, ETS, RUSH1A, TBP, YY1
Tongue ARF, CREB, CREBP1, ATF, NRF1, CEBP, CEBPA, SREBP1, MYOGNF1, VDR, HIC1, CP2, ZIC3
Uterus* E4BP4, POU1F1, POU3F2, NKX6-2, OCT1, AP1, HMGIY, ZIC3, PLZF, YY1, AIRE, PLZF, WT, SP3, CREB, NRF1, ELK1, ETF, SMAD-3, EF2

Table 1. This table contains information on the transcription factors that are active in different types of cells in the human body. It was generated based on published scientific studies (see the end of the Bibliography in the Background section for the citations).

* For the brain and uterus, there may be alternative, or additional, names listed for these tissues that you may need to find out in order to investigate them in the microarray database, which is explored in the section titled "How Specific Are Your Selected Transcription Factors?"

  1. In your lab notebook, copy and fill out Table 2 with the names of the important transcription factors listed in Table 1 with the tissue type that you selected. Confusingly, sometimes the same transcription factor can have multiple different names or abbreviations, or "aliases."
    1. If there are more than ten transcription factors listed for the tissue type you chose, just pick ten.
    2. Table 2 has been filled out using the kidney as an example, but you can fill it out with information on a different target cell type.
    3. You will fill out the other columns of Table 2 as you go through the Experimental Procedure.
Target Cell Type Important Transcription Factor Other Cell Types That Express It Highly Expressed in Other Cell Types? Known Function(s)
Kidney HNF1 Oocytes, liver, small intestine, PNS Yes (1 of 4) Associated with liver-specific genes. Defects can cause diabetes and liver tumors. Involved in diabetes and insulin signaling pathways.
Kidney HNF4    
Kidney CRX    
Kidney GCNF    
Kidney HNF3   
Kidney PPARA    
Kidney SP3   
Kidney ER    
Kidney TEL2   
Kidney SREBP   

Table 2. This table contains information on transcription factors that are important for kidney cells, as an example. For the first transcription factor, HNF1, all of the relevant information has been entered. For the next nine transcription factors, fill in the information in the empty cells as you go through the Experimental Procedure. Alternatively, choose your own target tissue from Table 1 and fill in Table 2 for that tissue.

How Specific Are Your Selected Transcription Factors?

You now have a list of transcription factors known to be active in your target cell type, but how specific are they? Are they active in a lot of other cell types too? In order to directly reprogram cells efficiently to your target cell type, the transcription factors used should be fairly specific to your target cell type. Alternatively, you could try to find a combination of transcription factors that, taken together, are uniquely active in your target cell type. Researchers often do this by using three or four transcription factors together. In this part of the science project, you will look at how your selected transcription factors are expressed (turned into mRNA and thus likely into protein) in a wide range of tissue types.

  1. You can learn about the expression of your selected transcription factors of interest by using Amazonia!, a database of microarray data. A microarray is a tool that enables researchers to look at a certain group of cells or tissues and see what genes are being expressed.
    1. At the bottom of the main Amazonia! page, there is a search box next to where it says "QuickStart: Explore the expression profile of any gene." In the search box enter the name of one of the transcription factors you wrote down in Table 2 and click "Go!"
      1. For example, for kidney search for HNF1.
    2. Under the section titled "Result(s) for Gene & Aliases" click on the blue link for your selected transcription factor. If there are multiple results shown, click on the top result that has your selected transcription factor name, such as HNF1, in red.
      1. The name of your transcription factor might only be listed under "aliases." Look for the words "transcription factor" next to one of these results. If the entry you found goes by a very different name than the one you searched for, make a note of this in your lab notebook.
      2. Not all of the transcription factors listed in Table 1 may have microarray data on them in the Amazonia! database. If your search yields no results, skip that transcription factor and pick a different one from Table 1 for your tissue type.
    3. Clicking the blue link for your selected transcription factor will take you to a page with microarray data on the transcription factor. For the HNF1 example, there are four graphs of data on this page. Click on the top graph, as shown in Figure 1, circled in red.
      Amazonia! is a microarray database
Medical Biotechnology science project

      Figure 1. Amazonia! is a microarray database that has a variety of expression data for a given gene. When looking at a page with data on a transcription factor, clicking on a graph (circled in red) will enlarge the graph and enable you to see its amount of expression in different cells and tissues in the body.

    4. Click on the graph image to enlarge it. On the x-axis (the horizontal axis) there are cell types listed. ("HESC" and "HIPS" are stem cells and can be ignored for this science project.) For every cell type, the levels of gene expression (labeled "microarray signal") are shown on the y-axis (the vertical axis).
    5. Find where your tissue type of interest is on the x-axis. For example, for HNF1 look for the word "Kidney." (Hint: The data for kidney samples are mostly in yellow on the first HNF1 graph.)
    6. Looking at the graph, does your target cell type express the highest amounts of your selected transcription factor of interest? Are there other target cell types that express the same amount or more of your transcription factor?
      1. If other tissue types look as if they express the transcription factor at a similar level to or at higher levels than your target cell type, do the following:
        • Go back to the page with microarray data on the transcription factor, as shown in Figure 1, and click on the symbol of the magnifying glass just under, and on the right side of, the graph you were looking at. This will open another window with all of the numerical data from the graph you were just looking at.
        • Under the "SAMPLES" column find your target cell type. Calculate the average of the "Signal" that is listed for your target cell type samples. For example, the average kidney signal is 166.7.
        • Calculate what 90% of the average signal for your target cell type is. For example, for the average kidney signal, this is 90% of 166.7, or 150.0.
        • Find the tissue types that looked like they express similar, or higher, levels of the transcription factor. If at least one quarter (such as one of four or three of 12) of the samples have values that are at least 90% of the average signal in your target cell type, write down the name of the tissue types in Table 2 in your lab notebook, under the column titled "Other Cell Types That Express It."
          1. In our example, you are looking for other tissues where one quarter of the samples have a signal of 150.0 or higher.
        • For example, according to the first graph, HNF1 is highly expressed in kidney cells, but it is expressed at similarly high levels in oocytes and liver cells.
      2. If you find a graph that has more than five different cell types that highly express the transcription factor, do not bother writing all of these tissue-type names in Table 2. Instead, write "Yes" under the column titled "Highly Expressed in Other Cell Types?"
        • This means that you found a graph that showed that the transcription factor in question is expressed at low levels compared with several other cell types. Consequently, the transcription factor might not be a good one to use to make your tissue of interest because it is not specific enough for your tissue. In other words, this transcription factor might make the cell type you want, but it might also make undesired cell types.
        • Keep track of how many graphs show relatively low expression of your selected transcription factor and write down this number out of the total number of graphs you looked at for the transcription factor. This has already been done in Table 2 for HNF1.
    7. Go back to the Amazonia! page with microarray data on the transcription factor of interest and scroll down to the next graph. Click on the graph and repeat steps 1d to 1f.
      1. Repeat this for each graph that is listed for a given transcription factor.
      2. For HNF1 for the kidney example, this information has already been filled out in Table 2.
  2. Repeat step 1 searching for the other transcription factors you selected for your target tissue.
    1. Reminder: You should have already written these transcription factors down in Table 2 in your lab notebook from the section titled "Finding Transcription Factors in Your Target Cell Type" (step 2).

Direct Reprogramming Concerns

Although some transcription factors look like they might work great at directly reprogramming cells to become a particular target cell type, it is important to make sure that these transcription factors will not cause problems for the cells. For example, some researchers used transcription factors that are associated with cancer to reprogram cells to become induced pluripotent stem cells (iPSCs). Although iPSCs are stem cells with great medical potential, because some are made using transcription factors associated with cancer, this has made people concerned about using them in medical applications. In this part of the science project, you will look at the normal function of your selected transcription factors.

  1. Learn as much as you can about each of your selected transcription factors. You can do this using the NCBI Gene Database.
    1. Use the Science Buddies NCBI Gene & SNP Tutorial to help you navigate the database. As an example, you can use the transcription factor HNF1 for your searches.
      1. Usually the transcription factor protein name will be the same as its gene name, so just use the name listed in Table 1 for all searches.
      2. Make sure to choose the top result that lists your transcription factor name, such as "HNF1," and is in humans (Homo sapiens).
    2. What does the database tell you about your selected transcription factor? Are there any diseases associated with it?
      1. Keep in mind that even if defects in the transcription factor's gene may cause diseases, this does not necessarily mean that making a cell produce the transcription factor will cause the same diseases.
    3. Add this information to the column titled "Known Function(s)" in Table 2 for each of your selected transcription factors. This has been done for HNF1 as an example.
  2. Learn about the signaling pathways (biochemical pathways) that each of your selected transcription factors are involved in by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database.
    1. In the search box under "Enter keywords," type in the name of a transcription factor and click "Go." For example, search for HNF1.
    2. Click on the "Thumbnail Image" for each pathway result.
    3. Look at the pathways for the transcription factor. It should be in red on the pathway diagrams, but if you cannot locate it, search for its name in the search box at the top of the page.
      1. What cellular processes is it involved in? What downstream events (events that the transcription factor leads to) might be triggered if a cell produces your selected transcription factor? Are any of these effects undesirable?
      2. To learn more about any protein in the pathway, click on its name (inside a box).
      3. Add information learned about the transcription factor to the column titled "Known Function(s)" in Table 2. As an example this has been done for HNF1.

Selecting the Best Transcription Factors for Direct Reprogramming

You have now learned a lot about your selected transcription factors. Table 2 should now be completely filled out in your lab notebook for your target cell type. Overall, how good to you think your selected transcription factors could be at directly reprogramming some cells into your target cell type? In this part of the science project, you will evaluate your different selected transcription factors and decide which would be the best - and the worst - to use in direct reprogramming experiments.

  1. How specific are the transcription factors for your target cell type? To answer this question, look at Table 2 in your lab notebook at the columns titled "Other Cell Types That Express It" and "Highly Expressed in Other Cell Types?" and think about the following questions: Are many of the selected transcription factors highly expressed in other cell types? If they are, is it usually the same cell type, or is it a wide range of cell types that they are expressed in?
    1. How might this affect your ability to directly reprogram cells to your target cell type using these transcription factors? How could these transcription factors turn cells into cells other than your target cell type?
    2. How might this affect the cell type you choose to start with, the starting material for your direct reprogramming experiments? If it is usually the same cell type that is also expressing your selected transcription factors, you may be able to use this to your advantage.
    3. Are there many graphs for your selected transcription factors that show low expression of your transcription factor in your selected cell type? How may this affect which transcription factors you choose for your direct reprogramming efforts?
    4. Look back at Table 1. Are any of your transcription factors listed as being active in other cell types? If so, does this agree or disagree with what you wrote down in Table 2 in the column titled "Other Cell Types That Express It"? How might this further affect your selection of transcription factors?
      1. Note: You can use Ctrl+F to find a word or specified text on this page.
  2. Do any of the transcription factors cause diseases? Look at Table 2 in your lab notebook at the column titled "Known Function" to answer this question. How does this affect which transcription factors you will select for direct reprogramming experiments?
  3. Overall, based on steps 2 and 3, evaluate which transcription factors may be the best ones to use in direct reprogramming efforts to make cells of your target cell type, and which ones may be the worst. You could narrow it down to about four transcription factors that may work well together. Also think about how this may affect which cell type(s) you would like to use as your starting material.

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Variations

  • In this science project, the focus is primarily on selecting the right transcription factors to create cells of your target cell type, without focusing much on what the starting cell material would be. However, some cells are more easily obtained than others, and consequently make a good starting material. Look at Table 1 above and think about which cells may be the easiest to obtain for research experiments. What target cell types could you most easily reprogram these fairly obtainable starting cells into? Find information on the transcription factors that are important for these cell types, as listed in Table 1, by repeating this science project. However, this time focus on thinking about how you can use it to your advantage when the expression of the selected transcription factors is similar to other cell types, particularly cell types that would make good target cell types.
  • Several different cell types use the same transcription factors, as you can see by looking at Table 1 above. Why do you think this is? Pick a few of the transcription factors that are important for multiple different cell types. Are the cell types that share a transcription factor related to each other, by function, their embryonic origin, or some other way?
    • You can read about the three different germ layers, which occur during early animal development and pattern the tissues and organs of the adult animal, to learn more about how different tissues and organs are related to each other. Here is a helpful website on this topic: http://biology.kenyon.edu/courses/biol114/Chap14/Chapter_14.html
  • Researchers have published scientific studies on many different kinds of direct reprogramming experiments. You can search for these papers and see how they selected their transcription factors. How was what they did different than what you did in this science project, and how was it similar? After selecting transcription factors that would work well in a direct reprogramming experiment, what are the next steps taken by scientists in a research lab? What kinds of problems do they encounter, and how do they overcome them?
  • Table 1 was generated from two different published scientific studies. However, these papers contain a lot more information on transcription factors than what was shown in Table 1 above. For example, transcription factors usually work by combining with other transcription factors. Look at these papers to find out which transcription factors in Table 1 interact with each other and think about how this could affect your selection of transcription factors for potential direct reprogramming experiments.
    • Here are the two papers:
    • Alternatively, you can explore transcription factor expression in cell types that are not listed in Table 1, but that are listed in these two papers. Look at the papers listed above to learn more.

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