BIO 202 MT 1

Antibodies may inhibit infection by

(a) preventing the antigen from binding to its target, (b) tagging a pathogen for destruction by macrophages or neutrophils (c) activating the complement cascade.

Examples of Stem Cells

-Artificial skin from umbilical cord stem cells -Bone stem cells shown to regenerate bone and cartilage -Stem cells from dental pulp -Bone marrow from hip, inject into damaged heart, stem cells embed themselves and produce proteins for growth signaling of new heart muscle and blood vessels -UMBLILCAL CORD STEM CELLS = immunodeficiencies, leukemia, myelomas, lymphoma, etc

Stem Cell Applications - why are they valuable

1) Help to understand basic genetic mechanisms that underlie process of multicellular development 2) Potential to treat human diseases or injuries -Tissue repair (bone, nerve, heart, muscle, skin, organ) -Autoimmune diseases (diabetes, rheumatoid arthritis, MS)

Types of Vaccines

1) Whole Organism -Live (attenuated) Viral/Bacterial -Killed (inactivated) Viral/Bacterial 2) Subunit vaccines 3) DNA based 4) RNA based (NEW)

Transcription

1. Initiation •Promoter : "start" signal; a specific binding site for RNA polymerase. •RNA polymerase : expose ssDNA. 2. Elongation •RNA elongates and peels away from its DNA template. 3. Termination •RNA polymerase reaches a terminator. •mRNA or "transcript" exits the nucleus and enters the cytoplasm.

Insulin Production!!!

1. Isolate gene for human insulin 2. Insert insulin gene into bacteria A. Insert insulin gene into a DNA vector B. Insert recombinant DNA (vector + insulin gene) into bacteria 3. Use bacteria to produce insulin protein

Applications of Monoclonal Antibodies

1. Research 2. Diagnostic testing 3. Diagnostic imaging 4. Treatment

Biopharmaceuticals Applications

1. Therapy 2. Prevention 3. Diagnostics

Evolution of Biotechnology

1972 Cohen (Stanford) and Boyer (UCSF) Biotechnological Revolution; established recombinant DNA technology 1980 Diamond v. Chakrabarty genetically modified organisms can be patented 2009 FDA approves first drug produced in a genetically engineered animal ( ATryn )

DNA Vaccines

A DNA vaccine is composed of DNA that encodes antigens. After administration of the DNA, antigens are produced and stimulate an immune response. Uses only the DNA from infectious organisms Avoid the risk of using actual infectious organism Provide both Humoral (B cells and Abs) and Cell mediated (T cells ) immunity Refrigeration is not required

DNA Microarray

A technique used t o study simultaneously gene expression of ~1,000 genes Utilized a microarray chip , which is a rectangular chip with attached single stranded DNA molecules cDNA from an organism , which one is investigating, is fluorescently labeled

Adeno-Associated Virus Vector for Gene Therapy

Advantages for gene therapy: -No viral genes in vector (no immune response to viral proteins) -Gene expression lasts months to year -No integration into chromosome (no risk of cancer) -Works best in post mitotic cells such as heart, skeletal muscle, brain, retina, liver -Main limitation: pretty small vector (can only hold 3.6 kb of non viral DNA)

Retrovirus for Gene Therapy

Advantages: -RNA is reverse transcribed to DNA, which stably integrates into the genome -allows permanent gene transfer -high expression levels of proteins Disadvantage = can infect dividing cells

Biopharmaceuticals vs Chemical Drugs

Advantages: -high selectivity -low nonspecific toxicity Disadvantages: -high costs -more complex

Adenovirus Vector for Gene Therapy

Advantages: -infects most cells -can infect dividing and non dividing cells -high expression levels of proteins -does not integrate into chromosome (no risk of cancer)

Examples of green biotechnology

Agricultural: the use of genetically altered plants or animals development of transgenic plants manipulation and use of microorganisms to facilitate crop growth, producing fertile and resistant seeds, etc. Environmental: bioremediation development of biofuels and biofertilizers applications in geomicrobiology, microbial ecology, solid waste management, wastewater treatment, etc.

Agrobacterium inserts DNA into plant genome

Agrobacterium tumefaciens - soil bacteria used to infect plants to make GM plant (inserts foreign DNA) Put desired gene in Ti plasmid, put recombinant plasmid back in bacteria, inject into plant cells, bacteria infects plant and integrates into genome Genetically engineering plants using Ti plasmid

Antigen

Any foreign substance that elicits an immune response when introduced into the tissues of a susceptible animal Typically proteins or polysaccharides , also polypeptides, lipids, and nucleic acids function as antigens Microbes are antigenic

Vaccines

Are parts of a pathogen or whole organisms that can be given to humans or animals by mouth or by injection to stimulate immune system against infection by those pathogens. Are designed to be preventative and not therapeutic.

Increasing crop yield to feed growing population

As of 2015, 26 plant species have been genetically modified and approved for commercial release in US GM crops!

Monoclonal Antibodies

B cell is specifically stimulated by one particular antigen and is then allowed to proliferate and produce Abs Abs all have the same antigenic specificity

Bt bacteria that kills insect larva used as a pesticide

Bacillus thuringiensis (Bt) - biological pesticide Cry gene = crystal protein Plant produces crystal protein, larva eats plant, proteins poisons gut of insect larva, larva die Used for 50 years in farming crops controversy about monarch butterflies = GM plant with toxins, wind transfer pollen to milkweed with butterfly larva

Common source of stem cells

Bone marrow: (HSC and MSC) Found in spongy bone where blood cells form Invasive and painful procedure for the donor, requires a greater immunocompatibility Treat patients diagnosed with leukemia, aplastic anemia, and lymphomas

CRISPR/Cas9 Applications

Genome editing is of great interest in the prevention and treatment of human diseases Currently, most research on genome editing is done to understand diseases using cells and animal models. Scientists are still working to determine whether this approach is safe and effective for use in people.

Herbicides kill plants

Glyphosate = chemical in Roundup --> stops action of EPSP synthase and plant dies Engineered resistance to weed killers = Ti engineered plasmid with glyphosate insensitive EPSPS gene, BACTERIAL EPSP protein remains active after spraying glyphosate Issue = Weed populations becoming resistant to glyphosate -->Random mutation gives resistance to glyphosate, spraying kills most weeds except those that are resistant, now have high % of weeds that are resistant

Examples of transgenic animals

Herman the bull = Human gene lactoferrin --> iron binding protein for diarrhea, liver inflammation, anemia, blood infection EnviroPig = Bacterial gene phytase --> less phosphorous in feces, first GM food animal on market Milk of GM goats produces human anticoagulant anti-thrombin, first drug produced in GM animal Using animals as bioreactors = cellular system used to make biological molecules

Transgenic created by pronuclear microinjection

Inject DNA into pronucleus of fertilized egg Implant embryos into surrogate mother Screen newborns for gene of interest - encode GFP (green fluorescent protein) gene or take part of tail and isolate DNA and run PCR to recognize your gene Breed transgenic organisms

Subunit Vaccines

Inject fragment of a pathogen, which will stimulate an immune response Include only the antigens that best stimulate the immune system --> they produce a very strong immune response The chances of adverse reactions: LOW

Liposome Gene Delivery

Liposome/DNA complex is taken up the cell endocytosis In the nucleus, DNA is transcribed to mRNA DNA will not integrate into cells

Biopharmaceuticals

Medicinal and therapeutic drugs -They consist of sugars, proteins, nucleic acids (DNA and RNA), living cells, or tissues -the extraction from living systems (humans, animals) or the production by recombinant DNA technologies and use of microorganisms.

Killed/Inactivated Vaccine

Methods of inactivation include heat, chemical agents (e.g. formaldehyde), and radiation Loss of replication Difficult to inactivate due to potential for denaturation of epitopes (dependence on higher order levels of protein) requires multiple boosters, more stable (fridge, 3rd world countries) produces mainly humoral immunity cannot revert to virulent form

Genetic Testing

Molecular genetic tests (or gene tests ) study single genes or short lengths of DNA to identify variations or mutations that lead to a genetic disorder. Genetic testing can be used to: a. diagnose or rule out suspected genetic disorders, b. predict risks for specific conditions, c. gain information that can be used to customize medical treatments based on an individual's genetic makeup, d. to determine biological relatives through DNA paternity testing.

Diagnosis

Monoclonal antibodies can be applied in the diagnosis of some cancers and infectious diseases Once monoclonal antibodies specified for a given substance are produced, they can be used to detect the presence of this substance.

GM animals

Purpose: drug production, enhance yields, increase resistance to disease Process is slow and expensive a) You first isolate the gene you want to inset into the host organism b) You then combine the gene of interest with other genetic elements c) Lastly, you insert the isolated gene into the host genome (there are several different techniques to do it - pronuclear microinjection)

RNA Vaccines

RNA vaccines work by introducing an mRNA sequence coding for a disease specific antigen Once the mRNA strand in the vaccine is inside the body's cells, the cells use the genetic information to produce the antigen This antigen is then displayed on the cell surface, where it is recognized by the immune system Faster and cheaper to produce than traditional vaccines RNA vaccines can be delivered: via needle syringe injections; via injection into the blood, muscle, lymph node or directly into organs; or via a nasal spray CHALLENGES - Unintended effects: the mRNA strand in the vaccine may elicit an unintended immune reaction. Storage: many RNA vaccines, like conventional vaccines, need to be frozen or refrigerated.

Vaccines still need...

Search for unavailable effective vaccines for diseases like: malaria, AIDS, toxoplasmosis Improvement in safety and efficacy of present vaccines Low cost Efficient delivery to needy Reduction of adverse side effects Storage

Gene Therapy

Technique which utilizes the therapeutic delivery of nucleic acids into patient's cells as a drug to treat a disease caused by a faulty gene or genes. Critical: safe and effective delivery of therapeutic genes. Depending on the genetic condition to be treated, some therapeutic strategies may require long lasting expression of a corrective gene, whereas others may require rapid expression for shorter periods of time. Use of viruses as vectors

Cas9-RNA complex recognizes and cleaves target DNA (nucleases)

Three components: Cas9 crRNA tracrRNA crRNA guides Cas9 to matching viral DNA sequence Cas9 cleaves DNA Cas9 inactive + crRNA --> Cas9 complex active

A simplified version of Cas9 for genetic engineering

Two components: Cas9 Guide RNA (single RNA combining crRNA & tracrRNA) Link that combines crRNA and tracrRNA into synthetic single guide RNA (in lab!) Using cell's DNA repair mechanisms to edit DNA after a break (double stranded break repair mechs)

Prevention

VACCINES! It usually contains a biological agent that resembles a pathogen and is usually made from inactivated microbes, live attenuated microbes, toxoids (toxins), and or part of surface antigens (subunits).

Immune Response

Vaccines teach our immune system by mimicking a natural infection B cells = humoral response --> antibody production T cells = cell mediated response --> cytotoxic to kill cells

Active Immunization

When the host is exposed to the foreign challenge and the host's native immune cells generate specific immune products. Stimulate the proliferation of T and B cells, resulting in the formation of effector and memory cells. The formation of memory cells is the basis for the relatively permanent effects of vaccinations.

Genome Editing

a group of technologies that give scientists the ability to change an organism's DNA allow genetic material to be added, removed, or altered at particular locations in the genome an enzyme cuts the DNA at a specific sequence, and when this is repaired by the cell a change or 'edit' is made to the sequence

Chromosomal Genetic Testing

analyze whole chromosomes or long lengths of DNA to see if there are large genetic changes, such as an extra copy of a chromosome, that cause a genetic condition. ex prenatal testing

Therapy

are able to treat diseases safely and effectively by demonstrating biological activity and perform specific functions by acting on the disease pathophysiology

Mesenchymal stem cells (MSCs)

derived from bone marrow gives rise to connective tissue = bone, cartilage, fat

Hematopoietic stem cells (HSCs)

derived from bone marrow gives rise to different types blood cells - lymphocytes, erythrocytes, neutrophils, basophil, eosinophil, platelet

CRISPR/Cas System

discovered in bacteria as their adaptive immune response mechanism against foreign DNA such as viral DNA (viruses infect bacteria) The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps: 1) Adaptation - virus invades, new spacer derived and integrated in CRISPR sequence 2) Production of crRNA - CRISPR RNA is formed 3) Targeting - if the same virus infects the bacteria, CRISPR will recognize pathogen DNA and will destroy it (complementary seq, cut with nuclease) CRISPR are clustered genetic elements in the bacterial genome that contain parts of viral DNA ("spacers") that infected the bacteria acquired from the past viral infections. Each spacer from a prior exposure from a different pathogens Only transcribed to RNA, not translated = final product of CRISPR is RNA

Genetic Disorder

disease caused by an abnormality in an individual's DNA 1) Single gene disorders involve mutations in the DNA sequence of a single gene; Examples: cystic fibrosis, sickle cell disease, Huntington's disease 2) Multifactorial disorders involve variations in multiple genes, often coupled with environmental factors; Examples: Alzheimer's disease, breast cancer

Red biotechnology

health and medical sector including diseases & diagnostics The use of living cells and cell materials to research and produce pharmaceutical and diagnostic products that help treat and prevent human diseases. Major Divisions 1) Biopharmaceuticals 2) Genetic testing 3) Gene therapy 4) Pharmacogenomics

Potency

how many different cell types it can change into, high potency = can change into lots of different cells unipotent - changes to the specific type of cell (red, white, etc) multipotent - specific tissues/lineage (hematopoietic) pluripotent - all except placenta totipotent - all

Neural stem cells (NSCs)

in the brain gives rise to specialized cells = neurons, oligodendrocytes, etc

White/Grey Biotechnology

indicates the industrial sector

iPS cells

induced pluripotent stem cells add factors to force specific differentiation transcription factors

In vivo gene therapy

inside the body - put gene of interest into vector, inject vector into patient issue = how to target intended tissue

Blue Biotechnology

is for aquaculture and marine

Selective Breeding

mating organisms with desired features to produce offspring with the same characteristics enhancing desired traits Advantages: Used for centuries, accepted by public Easier to select for traits controlled by multiple genes Can be used without knowing which genes are responsible Disadvantages: Slow process, less precise than GE Limited to the traits found within that species

Ex vivo gene therapy

outside the body - remove cells from patient, grow cells in culture, insert gene, inject back into patient

Transgenic Fish

production much easier than making other transgenic mammals Fish produce a large number of eggs can generate large quantities of genetically uniform material for experimentation 1st success 1985 microinjected the human GH gene into the fertilized eggs of goldfish GloFish = first commercialized GM animal --> green fluorescent protein (GFP) from jellyfish AquaAdvantage Salmon = approved for food --> carries growth hormone year round, reaches market size in 18 months vs 3 years

Green Biotechnology

refers to the agriculture or food, plant and environment Agricultural biotechnology involves the use of scientific tools and techniques to develop the agricultural sector Environmental biotechnology focuses on using microorganisms to prevent, reduce, and treat environmental pollution.

Stem Cell (regenerative medicine)

self-renewal commitment = differentiation (can make different types of classes)

Molecular Genetic Testing

study single genes or short lengths of DNA to identify variations or mutations that lead to a genetic disorder. I. diagnostic testing II. preimplantation genetic diagnosis

Biochemical Genetic Testing

study the amount or activity level of proteins ; abnormalities in either can indicate changes to the DNA that result in a genetic disorder. ex newborn screening

Ti plasmid (tumor inducing)

tDNA = promoter and gene of interest gene of interest - tDNA, inserted into the plant and integrated into plant genome promoter - DNA sequence that defines transcription of a gene by RNA pol virulence genes kept intact - encode proteins that help infect the plants, is not inserted into the plant antibiotic resistance gene - helps to select transgenic bacteria, grow on plate and see which survive low efficiency of actually being incorporated

Biotechnology

using living organisms, or the products of living organisms, for human benefit to make a product or to solve a problem.

Attenuated Vaccine

weakened It is achieved by growth under abnormal culture conditions to prevent produces humoral and cell mediated immunity ADVANTAGES: -Prolonged immune system exposure -Single immunization DISADVANTAGES: -Possible reversion and regaining disease causing properties -Inactivation may be incomplete -Unforeseen postvaccine complications

Gene transfer from GMO crops

wind transfers pollen from GM plant to non GM plant, only bacterial genes transferred fragments of genes enter organs of humans New federal law requires labeling food from GMOs (text, symbol, QR or code) = Obama 2016