Introduction to Genetic Engineering Transcript PDF

PROPRIETARY. DO NOT SHARE. Transcript: Introduction to Genetic Engineering Section 1: DNA and Protein Review Welcome Welcome to Introduction to Genetic Engineering. In this course, we will discuss how cells can be used to create a protein of interest. These proteins can then be used as therapeutics...

PROPRIETARY. DO NOT SHARE. Transcript: Introduction to Genetic Engineering Section 1: DNA and Protein Review Welcome Welcome to Introduction to Genetic Engineering. In this course, we will discuss how cells can be used to create a protein of interest. These proteins can then be used as therapeutics for various disease states. This course will be broken into 3 sections. The first section of this course will focus on reviewing some information about DNA and how it is used inside the cell to create proteins. This section will only provide a short review, if you would like a detailed explanation of DNA and proteins, please refer to the Biology of Biotech course. The second section will introduce the concept of Recombinant DNA. Lastly, the third section will discuss how Recombinant DNA is inserted into a cell to create recombinant proteins. Section 1: DNA and Protein Review Objectives Section 1 will serve as a short review of DNA and proteins. ● First, we will remind you of the structure of DNA. ● Next, we will illustrate how cells can convert specific portions of DNA, known as genes, into proteins. This cell machinery will be important when using cells to create recombinant proteins. ● Lastly, we will give examples of some of the protein types and their functions. These examples will be important in section 3 of this course when we discuss the types of recombinant proteins that can be created and their uses in biotech. Cells Contain DNA All cells contain DNA; from unicellular organisms such as bacteria and yeast to much more complicated multicellular organisms such as plants, to the exquisitely complicated organism, the human body. Because DNA is present in all cells, we know that DNA is very important to the function of a cell. Deoxyribonucleic Acid (DNA) DNA is just a chemical. Of specific note here is that regardless of the organism, the structure of the DNA is the same. In other words, DNA is the same molecule whether it is in a bacterium or a 1 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. human body. This will be important when creating recombinant DNA and recombinant proteins because it means that bacteria can read and understand human DNA. Deoxyribonucleic Acid Structure DNA is a polymer or a chain of interconnected building blocks. The building block of DNA is called a nucleotide. A nucleotide is composed of a sugar known as deoxyribose, a phosphate group, and a base. Every single nucleotide has the same sugar and phosphate group, the differences are created by the component of DNA called the base. The base is where the information is contained in a DNA molecule. There are four bases. These bases are guanine, adenine, thymine, and cytosine represented by the letters G, A, T, or C. DNA Sequence DNA is a polymer composed of two strands of nucleotides with connections between the bases of each of the DNA strands. The base G always binds to C, and the base A always binds to T. When we talk about a DNA sequence what we are talking about is the order of the bases attached to the nucleotides in the DNA molecule. The sequence of these bases is what’s important when genetic information is used to create proteins. For more information on how DNA is assembled or replicated in the cell please refer to The Biology of Biotech course. Cells Receive Signals and Make Proteins To summarize, the process of the cell creating proteins known as gene expression starts with an external signal that occurs when a ligand binds with a receptor on the cell’s surface. To see a close-up of the receptor, click on the receptor on the cell, it is denoted by the blue arrow. Proteins Are Made by Cells This slide simply reviews the entire process of a cell creating a protein from DNA. For full explanations of the processes of transcription or translation please refer to Biotech 101. For the purposes of this course, it is simply important to know that this protein-making machinery inside the cell is what is used to create recombinant proteins discussed in section 3 of this course. 2 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Post-Translational Modifications Some proteins need to be modified with additional molecules to be fully functional. The most common examples of this are phosphorylation or the addition of a phosphoryl group (shown on the left) and glycosylation or the addition of a glycan group to a protein. Whether a protein requires these post-translational modifications to function will be important when discussing the cell type to use to create a recombinant protein. This will be discussed indepth in section 3 of this course. What Are Proteins? Lastly, this screen reminds us of a few different types of proteins. These different protein types have different structures which determine their functions inside or outside of the cell. When creating recombinant proteins, it will be extremely important to know the shape, complexity (i.e. does it need post-translational modifications, etc.), and function of the protein that we are trying to create. This will be discussed further in section 3. For now, it is enough to know that there are many different types of proteins, and proteins are really the workhorses of the cell. Therefore, the ability to create proteins of interest using recombinant technology has wide-reaching implications in healthcare as well as other biotech industries. Section 1: DNA And Protein Review Summary To summarize section 1: ● We first reviewed the structure of DNA by discussing the chemical structure of the building blocks of DNA, nucleotides. ● We then reviewed the process in which DNA is transcribed to RNA and then this RNA is translated to proteins. ● Lastly, we discussed a few types of proteins: enzymes, receptors, structural proteins, and antibodies, and that the shape of these proteins directly leads to the function of the protein. This review will be important when discussing the creation of recombinant DNA and recombinant proteins in sections 2 and 3 of this course. 3 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 2: Recombinant DNA Welcome In section 2 of this course, we will discuss how recombinant DNA can be created by first cutting out a gene of interest from DNA and then incorporating that piece of DNA into a structure known as a plasmid that can easily be transferred into cells. This information will transition into section 3 when we discuss how these plasmids can be used to create recombinant proteins inside these cells. Section 2: Recombinant DNA Objectives When discussing Recombinant DNA: ● We will first need to define what recombinant DNA is. ● After this, we will explain the steps used to make recombinant DNA. ● Lastly, we will describe some of the uses of recombinant DNA. Recombinant DNA Recombinant DNA is defined as DNA molecules that have been created by combining DNA from more than one source. The general strategy of genetic engineering is to: 1. Cut a human gene out of a human cell’s DNA. 2. Package this human gene with DNA (recombine it into a package called a plasmid). 3. Add this packaged human gene to a non-human cell (bacterial or mammalian) so that the cell now makes the human protein product specified by that gene. The specifics of these steps will be discussed over the next few screens. Therefore, we first need to discuss how the gene of interest is cut from the DNA. This is done with restriction enzymes. Restriction Enzymes Restriction enzymes are enzymes made by bacteria as part of their immune system to chop up any foreign DNA that they encounter such as viral DNA. These restriction enzymes can be utilized in Biotech to cut DNA into small pieces so that a gene of interest can be put into a different cell. There are hundreds of different restriction enzymes and each restriction enzyme cuts DNA at a different place. On this screen, we first illustrate a common restriction enzyme known as EcoR1. 4 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. EcoR1 comes from E. coli and recognizes a specific DNA sequence, in this case, GAATTC. So, any place that GAATTC is seen by the enzyme it will cut the DNA at that spot. Therefore, if the gene that we are interested in is located between two sequences of GAATTC then when EcoR1 cuts the DNA at these two places the gene of interest will be present in this smaller DNA fragment. Similarly, SMA1 recognizes CCCGGG. These different enzymes give us the ability to cut DNA at different sequences with reproducible and predictable cuts. Using this information, we can determine what enzyme can be used to make cuts around our gene of interest, creating a DNA fragment that contains that gene. That is the first step towards making recombinant DNA. Plasmids The next step is to place the gene of interest that we have isolated using restriction enzymes into a structure that we can introduce into a cell of interest. The most common of these structures is known as a plasmid. A plasmid is a short, circular piece of DNA that is easily transferred between cells. Plasmids occur naturally in bacteria and have now been engineered with many different characteristics depending on the gene that we are working with and the cell system we would like to transfer that gene into. This screen shows that a plasmid can enter a bacterial cell, carrying the gene of interest with it. A plasmid can also be used to introduce a new gene into mammalian cells, which will be discussed in more detail in Section 3. For now, all we need to know is that our human gene of interest can be integrated into the plasmid and then the plasmid can be transferred to the cells we wish to use. The next screen will show how restriction enzymes can be used to integrate the gene of interest into this plasmid. Combining The Gene of Interest with a Plasmid Recall that restriction enzyme cut at a specific sequence of bases. In the case of EcoR1, this specific sequence is GAATTC. So, if we add EcoR1 to a plasmid DNA it will cut that plasmid in the same way that it cuts the gene of interest. Once the plasmid has been cut, because the gene of interest will have ended with the same sequence of bases as the plasmid, these base pairs will be able to reconnect with the cuts in the plasmid because they are complementary. In this way, the gene of interest has now been integrated into the plasmid, the plasmid has now been recombined with our gene of interest. Let’s look at this process in a bit more detail. 5 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Making a Recombinant Plasmid On the right side of the screen, we have human DNA and this DNA contains our gene of interest. On the left, we have the plasmid that has been engineered for our purposes. We then add the restriction enzyme EcoR1 to both the plasmid and the human DNA. Because of the design of the plasmid, EcoR1 will create a gap in the plasmid, and similarly, the human DNA will be cut at the same sequence, and between these 2 cuts will be the gene of interest. Now we will simply put these two pieces of DNA in the same solution and the DNA will match up with each other due to complementary base pairing. An enzyme known as DNA ligase is then added to the solution to seal the plasmid ensuring that the plasmid now contains our human gene of interest. This plasmid is now known as a recombinant plasmid as it has been recombined with the human gene. The next step will be to put this recombined plasmid into a cell and use that cell’s machinery to create a recombinant protein, this will be discussed in section 3. Section 2: Recombinant DNA Summary To summarize section 2: ● We first defined recombinant DNA as DNA molecules that have been created by combining DNA from more than one source. ● We then demonstrated how recombinant DNA is created. The process involves cutting a portion of DNA out of a structure known as a plasmid and replacing this portion of the plasmid with a gene of interest. This process allows us to introduce human genes into other cell types such as bacteria or other mammalian cells. ● Lastly, we discussed that the reason for creating recombinant DNA is to introduce that DNA into a cell that can then create the protein coded for by that gene. The detailed process of using cells to create these recombinant proteins is the focus of section 3. 6 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 3: Recombinant Proteins Welcome Now that we know how recombinant DNA is created, in section 3 of this course, we will discuss how this recombinant DNA can be introduced into different cell types in order to have those cells create recombinant proteins. These proteins can then be used in healthcare as treatments or can be used for many other purposes in the field of biotech. Section 3: Recombinant Protein Objectives Section 3 will start by: ● Explaining how recombinant DNA is used to produce recombinant proteins. ● Next, we will compare and contrast the use of bacterial vs mammalian cells for creating recombinant proteins. We will see that both systems have their pros and cons. ● Lastly, we will describe some of the different uses of recombinant proteins in healthcare as well as other fields of biotech. Recombinant Proteins Recombinant Proteins are proteins that are made using the machinery of the cell to express a desired gene. In section 2 of this course, we demonstrated how recombinant DNA can be created and packaged into a structure called a plasmid. Once a recombinant plasmid has been created the next step is to put the plasmid into a cell and have that cell express our gene of interest. The most common cell systems used to create recombinant proteins are bacterial cells and mammalian cells. The process for using these two cell types to create our gene of interest will be discussed on the next two screens. Making Recombinant Proteins in Bacterial Cells Once we have a plasmid the next step is to transfer this plasmid into a cell of our choice. We will first discuss the use of bacterial cells to create proteins. Another thing to note here is that the plasmid also contains a gene that is able to make the cell resistant to antibiotics. This will be important once the plasmid has been put into the bacterial cell. There are a few methods for putting the plasmid into bacterial cells. Two common methods used when working with bacterial cells are heat shock and electroporation. Heat shock involves 7 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. suddenly increasing the temperature that the cells are exposed to in the presence of the chemical calcium chloride. Electroporation involves exposing the bacteria to short pulses of an intense electric field, a bit like striking the cells with lightning. In either method, the heat or the electric field creates pores in the membrane of the bacteria allowing the plasmid to enter the cell. It should be noted that not all bacteria will survive the process or take up the plasmid, so the next step is to get rid of all the bacterial cells that do not contain the plasmid. If we look here at a culture of bacteria, we can see that not all of the cells in this sample have taken up the plasmid, and the cells that do not contain the plasmid will not be able to create the recombinant proteins so they need to be removed from the culture so that the cells that contain the plasmid have space to multiply. This is where the antibiotic resistance gene that we mentioned earlier is used. The antibiotic resistance gene will allow the bacterial cells that have taken up the plasmid to create a protein that makes them resistant to a particular antibiotic. The other bacterial cells that have not taken up the plasmid will die in the presence of the antibiotic. Therefore, by simply adding the antibiotic to the cell culture we can kill the cells that have not taken up the plasmid, while at the same time selecting the cells that have taken up the plasmid to remain and multiply. All these selected cells will now be able to create the recombinant protein that our gene of interest codes for and release that protein into the cell media to be harvested and purified. Making Recombinant Proteins in Mammalian Cells This slide shows that a very similar process can be used to transfer recombinant DNA into mammalian cells. The reason for choosing bacterial cells or mammalian cells will be discussed on the next slide but first, we will discuss the process of using mammalian cells and how it differs from bacterial cells. Mammalian cells are more sensitive to environmental changes, and therefore will not survive the process of heat shock or electroporation that was used to transfer a plasmid into bacterial cells. Therefore, in this case, many times a structure known as a liposome is used. A liposome is a sac, often called a vesicle, into which we can inject the plasmid. The liposome’s membrane is composed of lipids just like the cell membrane of the CHO cells. Because of this, the liposome is able to enter the cell through the membrane. Once inside the cell, the plasmid is then released from the liposome and enters the CHO cell nucleus. Just like before when we talked about bacterial cells, in this case, not all the CHO cells will take up the plasmid, so we need a way to select only the cells that have taken up the plasmid. We 8 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. cannot use antibiotic selection in this case, because of course, all the mammalian CHO cells would be resistant to antibiotics. Therefore, in this case, we will simply add a gene for an essential nutrient that the cell needs to live. This essential nutrient would normally be given to the cells in the cell media used to culture the cells. However, in this case, we will remove that essential nutrient from the cell culture media. Because this nutrient has been removed only cells that can produce that nutrient themselves will be able to survive. In other words, only the cells that have taken up the plasmid can now produce the nutrient using the gene that we included in the plasmid. So, the result is that we will select only the cells that have taken up the plasmid, and again all these cells will now be able to create the recombinant protein that we are interested in creating. Again, this protein can now be harvested from the cell media and purified. The Factory: Bacterial vs Mammalian Cells Now that we have explained how recombinant DNA can be transferred to bacterial or mammalian cells, let’s explore the reasons that a researcher may choose one factory for creating recombinant proteins over another. The first thing to mention is that bacterial cells divide much more quickly than mammalian cells. A bacterial cell divides every 20 to 30 minutes while mammalian cells divide once a day on average. Because of this time difference, a full production campaign to create a recombinant protein in bacterial cells is completed in days rather than the weeks that a production campaign takes in mammalian cells. Additionally, the growth media for bacterial cells is relatively inexpensive when compared to the media needed to culture mammalian cells. Bacteria growth media costs around $40 per liter while media for mammalian cells costs around $100 per liter. While this seems like a small difference, remember that a production campaign could grow cells in a 20,000-liter vessel and that the media needs to be changed out during the process as well, so that adds up to a large difference in cost. To make the process of using mammalian cells even more difficult, mammalian cells are much more sensitive to changes in the cell culture environment such as temperature, pH, how tightly packed the cells are together, etc. Bacterial cells are less sensitive to these small changes in their environment. So, after looking at this list you might be wondering why anyone would want to use mammalian cells: they are slower, more costly, and more difficult to work with. And you would be right, if it was possible, it would always be easier and cheaper to use bacterial cells. 9 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. However, unfortunately, bacterial cells have one large flaw that makes it impossible for them to be used to create many proteins. Bacterial cells can only produce simple proteins, that is proteins that do not require complex folding of the protein or post-translational modifications to function. Bacterial cells are not capable of this more complex protein processing; however, mammalian cells are. Therefore, if the desired recombinant protein requires complex folding or posttranslational modifications, then a mammalian cell system must be used to create that protein. For a description of a protein type that requires a mammalian cell to manufacture the monoclonal antibody, click on the button to look closer. Otherwise, click next to continue the course. Monoclonal Antibodies The creation of monoclonal antibodies requires the use of mammalian cells because monoclonal antibodies must be glycosylated in specific locations in order to function in binding to their target. Since bacterial cells are unable to perform this post-translational glycosylation, mammalian cells are required. Recombinant Proteins in Healthcare Now that we understand how recombinant proteins are created let’s discuss a few examples of how these proteins are used in the Healthcare industry. This is by no means an exhaustive list, but it gives you an idea of the flexibility of the technology. The first and most simple way that this technology is used is to create proteins that might be absent or non-functioning in a person with a specific disease, and then use the recombinant protein to supplement or replace the absent or non-functioning protein in those patients. An example of this is the product ADVATE manufactured by Shire. This product is a recombinant form of the protein known as the antihemophilic factor, also known as Factor VIII. In patients with Hemophilia A, this protein is missing and therefore the patient’s blood will not clot correctly. By injecting ADVATE, the missing protein is replaced, allowing the patient’s blood to clot normally. The next application is the creation of vaccines. In this case, a recombinant vaccine for the human papillomavirus (HPV) known as GARDASIL was created by Merck. HPV has been estimated to cause about 70% of cervical cancers, therefore by giving the vaccine to women these cancers can be prevented. HPV has also been linked to other cancers such as vulvar and vaginal cancers in women as well as anal cancers in men, therefore the vaccination has also been approved for men. 10 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Yet another example of how recombinant technology can be used is to create monoclonal antibodies against autoimmune diseases or disease proteins. An example of this is the monoclonal antibody to the HER2 protein known as Herceptin produced by Genentech. The overexpression of the HER2 receptor has been shown to account for around 25% of breast cancers. This overexpression leads to uncontrolled growth of the breast tissue which leads to a malignant tumor. Herceptin blocks the activation of the HER2 receptor, thus stopping uncontrolled growth and slowing or even stopping tumor growth. More information on this mechanism can be found in the Biotech 101 course. These are just a few examples of how recombinant protein technology can be used in healthcare, if you would like to learn about other examples of how this technology is used in the biotech industry, click on the button on the right to look closer. If not, click next to continue. Best of Both Worlds: Fusion Proteins A new class of recombinant proteins is fusion proteins. These are proteins that combine the desirable characteristics of two different proteins. For example, the long-acting recombinant blood clotting factors Alprolix and Eloctate combine the blood clotting ability of a clotting factor with the stability of an antibody. Normally, blood clotting factors are not stable in a patient’s body, meaning that hemophiliacs receiving infusions of recombinant clotting factors need to be infused every other day. In contrast, antibodies are very stable, typically lasting for several weeks in a patient’s body. By fusing the gene for a blood clotting factor with a portion of the antibody gene that helps to confer stability – the antibody constant region – scientists have created these long-acting clotting factors. While not as stable as antibodies, they do allow less frequent administration of the clotting factors – twice a week rather than every 48 hours. Pharm Animals Most biologic drugs are produced in bacterial cells or CHO cells, as we have been discussing. However, a few companies are turning to alternative methods, such as genetically engineered animals, to produce biologic drugs. The principle is the same as what we have been discussing: to get an animal to produce a therapeutic protein, we must first transfer the gene for that protein to the cells of the animal. The resulting animal is called a transgenic animal. Typically, we would choose an animal that produces milk, such as a cow or a goat, and engineer the animal so that it only produces the therapeutic 11 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. protein in its milk. This is safer for the animal and makes it easier for us the harvest the protein – all we need to do is collect the milk and extract the therapeutic protein. How will we go about doing this? Let’s say we want to engineer a goat to produce protein in her milk. First, we’ll use restriction enzymes to isolate the gene of interest, as described before. We next make a piece of recombinant DNA consisting of the therapeutic gene and goat promoter DNA. Promoter DNA is DNA that is recognized by the enzymes that read DNA. Different promoter sequences help to turn genes “on” or “off” in different tissue types. Since we want to have the goat produce the protein only in her milk, we’ll use a promoter that is active only in udder tissue. The recombinant DNA is microinjected into the nucleus of a fertilized goat egg using a fine glass needle. The injected egg is transplanted into a surrogate mother which gives birth to a transgenic goat, now expressing the target gene in her milk. The milk can be harvested and the human protein of interest can be isolated by purification schemes. The protein shown is antithrombin which is an anti-clotting and anti-coagulation factor in the blood. Antithrombin from transgenic goat milk has been approved for certain thrombin deficiencies under the trade name ATryn (GTC Biotherapeutics). Pharm Plants Plants are also a possible production platform for biologic drugs. The type of plant most commonly used in this approach is tobacco plants. This is largely because we have a very efficient method of transferring genes to tobacco plants, in the form of the tobacco mosaic virus. This virus is extremely well-characterized, which means that scientists can easily engineer it to contain a therapeutic gene. The virus is then sprayed on tobacco plants, where they infect the plant cells, delivering the gene for the therapeutic protein. Once this gene is inside the plant cells, it is used to produce the specified protein. The final step is extracting the therapeutic protein from the tobacco leaves and purifying it. Currently, there are no FDA-approved biologics that have been produced in plants, but several are in development. 12 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 3: Recombinant Proteins Summary To summarize section 3: ● We first defined recombinant proteins as proteins that are made by using the machinery of a cell to express the desired gene. ● We then demonstrated that while using bacterial cells to create these proteins is easier, faster, and less expensive than using mammalian cells, sometimes mammalian cell systems are needed if the recombinant protein that we want to create is complex. ● Lastly, we gave examples of protein replacement, vaccines, and antibodies as just a few ways that recombinant proteins can be utilized in the field of healthcare. 13 Copyright 2023 Biotech Primer, Inc.