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Chapter 21-Transgenic Animals: Methodology and Applications

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Chapter 21-Transgenic Animals: Methodology and Applications

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    1. Chapter 21-Transgenic Animals: Methodology and Applications Transgenic mice: methodology (Retrovirus vector, DNA microinjection, Engineered embryonic stem cell, Cre-loxP recombination system, High capacity vectors) Transgenic mice: applications (Alzheimer disease, test systems, conditional regulation, control of cell death) Cloning livestock by nuclear transfer Transgenic cattle, sheep, goats and pigs Transgenic birds Transgenic fish

    2. Fig. 21.1 Establishing transgenic mice with retroviral vectors (rarely used)

    3. Fig. 21.3 Establishing transgenic mice by DNA microinjection Most commonly used method Only 5% or less of the treated eggs become transgenic progeny Need to check mouse pups for DNA (by PCR or Southerns), RNA (by northerns or RT-PCR), and protein (by western or by some specific assay method) Expression will vary in transgenic offspring: due to position effect and copy number

    4. Creating a transgenic mouse using the DNA microinjection method See http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00020&n=09000&i=09020.02&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=538 See also http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=09000&i=09010.10&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=661 And for reporter constructs, see http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=15000&i=15010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1322

    5. Establishing transgenic animals using engineered embryonic stem (ES) cells But what are ES cells?

    6. Transgenic animals-Engineered embyronic stem cell method (used for gene knockouts) Step 1: Get the ES cells (Fig. 21.5)

    7. Step 2: Genetically engineer the ES cells (Figs. 21.5 and 21.6)

    8. Step 3: Place engineered ES cells into an early embryo (Fig. 21.5) see http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00020&n=09000&i=09020.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=486

    9. Transgenic animals-Using Cre-loxP for tissue or time-specific gene knockouts

    10. Transgenic mice can be produced with high capacity vectors Generally done by microinjection of numerous genes contained in a YAC Production of mice that can produce human antibodies is one notable example

    11. Transgenic mice/animal: applications Transgenic models for Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, arthritis, muscular dystrophy, tumorigenesis, hypertension, neurodegenerative disorders, endocrinological dysfunction, coronary disease, etc. Using transgenic mice as test systems (e.g., protein [CFTR] secretion into milk, protection against mastitis caused by Staphylococcus aureus using a modified lysostaphin gene) Conditional regulation of gene expression (tetracycline-inducible system in Fig. 21.19) Conditional control of cell death (used to model and study organ failure; involves the organ-specific engineering of a toxin receptor into the mice and then addition of the toxin to kill that organ)

    12. Another Transgenic mouse application: Marathon Mice

    13. And then there is “transgenic art” with GFP…

    14. Fig. 21.22 Cloning livestock by nuclear transfer (e.g., sheep) “Hello Dolly”

    15. And now there is pet cloning for a “small” fee…

    16. Transgenic cattle, sheep, goats, and pigs Using the mammary gland as a bioreactor (see adjacent figure) Increase casein content in milk Express lactase in milk (to remove lactose) Resistance to bacterial, viral, and parasitic diseases Reduce phosphorous excretion

    17. Table 21.2 Some human proteins expressed in the mammary glands of transgenic animals Erythropoietin Factor IX Factor VIII Fibrinogen Growth hormone Hemoglobin Insulin Monoclonal antibodies Tissue plasminogen activator (TPA) a1-antitrypsin Antithrombin III (the first transgenic animal drug, an anticlotting protein, approved by the FDA in 2009)

    18. “Enviropigs” Transgenic pigs expressing the phytase gene in their salivary glands The phytase gene was introduced via DNA microinjection and used the parotid secretory protein promoter to specifically drive expression in the salivary glands Phytate is the predominant storage form of phosphorus in plant-based animal feeds (e.g., soybean meal) Pigs and poultry cannot digest phytate and consequently excrete large amounts of phosphorus “Enviro-pigs” excrete 75% less phosphorus Microinjected an E. coli phytase gene under the control of a mouse parotid secretory protein promoter

    19. Fig. 21.32 Establishing transgenic chickens by transfection of isolated blastoderm cells Resistance to viral, bacterial, and coccidial diseases Better feed efficiency Lower fat and cholesterol levels in eggs Better meat quality Eggs with pharmaceutical proteins in them

    20. Transgenic fish Genes are introduced into fertilized eggs by DNA microinjection or electroporation No need to implant the embryo; development is external Genetically engineered for more rapid growth using the growth hormone gene (salmon, trout, catfish, tuna, etc.) Genetically engineered for greater disease resistance Genetically engineered to serve as a biosensor for water pollution Genetically engineered for a novel pet (Glofish-see http://glofish.com/)

    21. Transgenic fish (more detail) Salmon were genetically engineered for more rapid growth using the growth hormone gene under the control of the ocean pout antifreeze protein gene promoter and 3’ untranslated region (currently under FDA consideration) Madaka fish were genetically engineered to serve as biosensors for environmental pollutants (e.g., estrogens) by using an estrogen-inducible promoter (the vitellogenin promoter) to control expression of the GFP gene

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