Genetics 2316 Exam 4

In RE analysis - what does one large fragment mean? Two small? One large two small?

1 large - affected 2 small - normal 1 large 2 small - heterozygous, but normal

4 areas ELSI focuses on?

1-Privacy 2-Ways to transfer knowledge to practice 3-Ensuring participants know the risks 4-Public and professional education

What are the two essential criteria for gene therapy?

1. Gene or genes involved in causing disease have been identified 2. Gene can be cloned or synthesized in lab

Success rate in T-DNA integration in creating GMOs?

1/1000

How many genetic diseases are identified per year?

12.5k/4000k

Gardasil vaccine?

4 strains of HPV - 6, 11, 16, 18 - which is the cause of 70% of cervical cancer

How many types of genetic tests? Examples?

900 Sickle cell, CF, Huntington's, breast cancer, colon cancer, can also identify in vitro embryos diseases and check for biparental inheritance

What are DNA profiles?

A forensic technique used to identify individuals by characteristics of their DNA such as minisatellites or Variable Number Tandem Repeats.

How are DNA samples obtained prenatally?

Amniocentesis - culturing and testing amniotic fluid cells Chorionic Villus Sampling (CVS) - testing of tiny finger-shaped growths in placenta that are genetically identical to fetus

RNA-based therapeutics

Antisense RNAs, RNAi, microRNAs for gene therapy

Example of a bioreactor and how does it work?

Antithrombin in goats which is used to help blood clotting disease. Gene is placed next to common milk protein so when promoter turns on milk gene it also leads to antithrombin production

2 methods of creating GM plants?

Biolistic Method - Gene gun w/ heavy metals that transform plant cells Ti Plasmid method - R. radiobacter (agrobacterium tumefaciens)

How is resistance to Glyphosate introduced to plants?

Biolistic method Agrobacterium CP4 EPSPS gene is cloned which is Glyph resistant, this gene is placed downstream of a constitutive promoter (in cytomegalovirus) so it is expressed in all cells. Chloroplast transit peptide is cloned to the 5' end so it could integrate into chloroplasts Treated w/ glyphosate to confirm transformation GUS gene from E. Coli is the marker (blue precipitate) - removed in final cell line

Benefits of transgenic animals?

Can increase size, protect against pathogens, produce eukaryotic proteins

3 disadvantages to using prokaryotic hosts?

Cannot process and modify eukaryotic proteins Cannot add side groups required for activity Do not fold properly

3 examples of Transgenic Animals and their uses?

Cattle - lack Prp gene helps resistance to prion disease EviroPig - reduce phosphorous excretion GloFish - contain fluorescent protein from sea anemones near their metal inducible promoter so they help detect the presence of metal

Nonviral delivery method for gene therapy

Chemically assisted transfer of genes across membranes. Nano particle delivery. (fusion of cells will artificial lipid vesicles) lipsomes

Lipsomes

Closed lipid bilayer spheres that encapsulate ingredients, target their delivery to specific areas of the skin, and control their release.

Edible vaccines? Ex?

Currently in clinical trials - produced in food plants Cholera vaccine in potatoes

Contents of DNA-based vaccines?

DNA encoding proteins from pathogen are inserted into vector and injected into an individual

DNA microarrays testing?

DNA fragments, similar to ASOs, are collected - difference is it can identify mutations that are larger and are more than 1 sequence long

What are the ways that therapeutic genes can be delivered into cells?

Delivery associated with chemical assistance to cross cell membranes, nanoparticles, or cell fusion with artificial vesicles. Therapeutic DNADNA are delivered into the cell by genetically engineered viruses.

What is Pharmacogenomics? Ex?

Designed to lead to more specific/effective/customized drugs determined by each person's individual genetic makeup. Ex: Thiopurine drug for childhood lueukemias, needs different dosages based on sequence variations in thiopurine methyltransferase Normal TPMT enzyme - metabolize quick (high dose) Normal and mutant - metabolize moderately Mutant enzyme - metabolize slow (low dose)

What are some of the major challenges that must be overcome to develop safer and more effective viral vectors for gene therapy?

Disabled viruses could recombine with an unmodified virus and regain their full infectivity, Regulation of the expression of the introduced DNA, An immune response to the introduction of viruses, Insertion in active genes can lead to unexpected interruptions of normal host gene functions.

What type of bonds join the A and B subunits of insulin?

Disulfide

How may the gene-silencing techniques be used for gene therapy?

Double-stranded RNARNA molecules are delivered into cells and are cleaved into short pieces of RNARNA (siRNAsiRNA). siRNAsiRNA can form a complex with enzymes that target mRNAmRNA, thus leading to its degradation. The silencing of deleterious or overactive genes may help alleviate disease symptoms, at least on a temporary basis. The antisense RNARNA binds to its complementary mRNAmRNA strand, thus blocking its translation.

Benefits/Use of transgenic plants?

Easily grown and are a good source of recombinant proteins (not yet approved by FDA, however)

What is ELSI?

Ethical, Legal, and Social Implications - program that ensures personal genetic information will be safeguarded and not used in discriminatory ways as well as the information's impact and implications on the lives of those who receive it.

What are the two approaches for deliver of therapeutic genes?

Ex vivo gene therapy and In vivo gene therapy

Transgenic salmon?

Express HGH and grow 10x bigger

What is Baculovirus?

Gene delivery system in which a virus is used to infect insect cells

Gene therapy?

Genes transferred into cells to treat disorders (normal alleles)

Restriction enzyme analysis? Ex?

Genetic test that analyses REs. Sickle Cell Anemia, RE = MstI

Allele-specific oligonucleotides testing?

Genetic testing where short, ss probes of DNA to identify alleles that differ by as little as a single nucleotide

Example of transgenic crops for herbicide and pesticide resistance? How was it created?

Glyphosate (round up) resistant maize and soybeans Gene inserted into Ti plasmid - R. radiobacter transfer of the recombinant vector

Golden Rice 2 contents?

Golden Rice 2 is a rice variety designed to synthesize beta-carotene. Golden Rice 2 involved the introduction of three genes into the T-DNA region of a Ti plasmid.

Example of nutritional enhancement of crops? How/why?

Golden rice Golden rice 1 - gene from daffodil and bacterium w/ moderate levels of beta-carotene Golden rice 2 - gene from maize with 20 fold increase of beta-carotene Helped areas in africa and asia with vitamin A deficiencies leading to blindness

What is the main challenge of RNAi-based therapeutics?

How to deliver dsRNA or siRNA. RNAs degrade quickly in the body.

2 important questions for edible vaccines?

How will dosage be controlled? WIll the vaccine pass through the digestive tract unaltered?

How synthetic human insulin is created?

Humulin is produced in bacteria (E. coli) E coli is transformed, one with gene A insulin subunit and some with gene B. These are then extracted and purified. The A and B chains are cleaved with cyanogen bromide and then A and B are mixed to form active insulin.

Ethical question about gene therapy/dna sequencing?

If it is known Huntington's will develop around the age of 40, it should be kept private. If insurance companies are told this, they may refuse to insure the patient.

Most important advancements of agricultural biotechnology?

Improve growth and yield Increase nutrition Herbicide and pesticide resistant plants

When treating a person by gene therapy, is it necessary that the therapeutic gene becomes part of a chromosome (integration) when inserted into cells?

Integration of the therapeutic DNA into the host genome is advantageous in that more stability of the introduced DNA is enhanced, which provides long-term expression; however, the integration process itself may generate mutations and regulatory alterations.

Mode of action of Glyphosate?

Interferes with EPSPS enzyme which is necessary for plant synthesis of aromatic AAs Phen, Tyr, and Trp.

How was the Hep B vaccine created?

It is cloned/purified in yeast

Inactivated Vaccines? Attenuated Vaccines? Subunit Vaccines?

Killed samples - ex: rabies Live samples that cannot reproduce - ex: cholera One or more surface proteins from the virus/bacterium (Hepatitis B)

Arguments for GM food?

Long-term toxicity tests show no harm No obvious negative effects have arisen due to GMOs Selective breeding doesn't require extensive clinical trials so why should GMOs

Example of transgenic animals for pathogen protection?

Mastitis (Staph aureus in milk of cows) - incorporation of gene protein that destroys the Staph auerus bacterium

Arguments against GM food?

Most GMOs not eaten by humans, but by livestock (not tested) Removes plant proteins Lack of clinical trials - some short-term toxicity tests have been done with negative results

Is EPSPS in mammals?

No, they get AAs from diet

How are retroviruses used in gene therapy?

Normal gene needed is cloned into retrovirus and then implanted into patient - normal gene is then produced by the retrovirus mechanism within the body

What is a direct to consumer genetic test?

Online companies that will test your DNA for mutations/diseases. Not always accurate and physician involvement is not necessary, and it is not FDA regulated.

How are transformed cells verified to have the co-transformed gene that is desired?

PCR or gene specific primers

Who is Jesse Gelsinger?

Patient with OTC liver disease who died due to a Gene Therapy treatment causing a severe immune reaction. First 17 patients were successfull then his failed. First major blow to gene therapy.

What subunits are present on the insulin plasmids?

Promoter, lacZ, insulin A/B, and then the antibiotic resistance gene.

How would a scientist silence a defective gene?

RNA Interference - short ds RNA molecules are delivered into cells where the enzyme Dicer chops them into 21-25nt long pieces called small interfering RNAs. siRNAs then bind to RISC which transfers them to their target mRNA where they bind and prevent translation.

Gene therapy can be...

RNA based.

Examples of Biopharmaceutical products?

Recombinant proteins Microbes - wine, beer, and cheese Recombinant DNA such as antibiotic resistance Therapeutic proteins that treat disease

genome editing

Removal, correction, and/or replacement of mutated gene. Genome editing by CRISPR-Cas has shown great potentials for gene therapy.

First generation of gene therapy trials uses what? First gene therapy used for?

Retroviruses Severe combined immunodeficiency disease (SCID)

What are the commonly used viral vectors?

Retroviruses. Lentiviruses. Adeno-associated virus. Adenoviruses.

Examples of gene therapy being used today?

SCID, HIV, cancer, retinal cone function and day vision in dogs, Leber's congenital maurosis, beta-thalassemia

Genome scan testing?

Scanning thousands of genes with a probe containing a SNP to find a mutation - different probes can be used to analyze DNA for many alleles of diseases.

How are transformed cells identified?

Selectable markers Positive Selection - cells w/ pmi gene cultured with mannose as only carbon source Negative Selection - cells w/ antibiotic resistance cultured with antibiotic

Prior to genetic engineering how was food production enhanced? Ex?

Selective breeding Maize - breeding larger ears and kernals

What were the two patent applications filed from the J. Craig Venter institute?

Shotgun approach to DNA sequencing, clone-by-clone approach in HGP, smallest bacterial genome sequenced ~381 genes

Examples of ethical questions about genetic testing?

Should we test people for disorders that have no medical treatments? How can we protect the information revealed? How should parents/physicians use the information that can be found from prenatal testing such as a gene that leads to Parkinson's, etc.

How are DNA profiles tested?

Southern blotting and Gel Electrophoresis of target region

Recombinant T-cell receptors (TCRs)

Specifically recognize antigens in cancer cells

Chimeric Antigen Receptors

TCR receptor + Antibody Chimera.

During the first successful gene therapy trial in which Ashanti DeSilva was treated for SCID, did the therapeutic gene delivered to Ashanti replace the defective copy of the ADAADA gene?

Targeted gene therapy was not possible, so integration of the ADAADA gene into Ashanti's genome probably did not replace her defective gene.

GFP-anti FIV testing?

Testing of FIV (feline AIDS) - basic research where fluorescent protein that glowed green where the anti FIV gene was present

What is Biopharming?

The production of proteins in GM plants and animals

What is biotechnology?

The use of living organisms to create a product or a process that improve quality of live

Because of ethical concerns surrounding gene therapy, what are gene therapy trails restricted to?

They are restricted to using somatic cells as targets for gene transfer.

Golden Rice 2 genes introduced how? Marker? Confirmation of transformation?

Ti plasmid pmi marker - mannose culture PCR for confirmation of transformation

What is the purpose of sequencing DNA in blood?

To analyze fetal haplotypes (Contiguous segments of DNA that do not undergo recombination). This helps distinguish which DNA segments are maternal and which are fetal.

Importance of Pharmacogenomics w/ TPMT?

Too little dosage could cause relapse while too much dosage could be fatal

Hosts for production of biopharmaceuticals?

Transgenic bacteria, plants, yeast, animals

What is a bioreactor?

Transgenic farm animals - form biofactories

What is the ultimate goal of translational medicine?

Translating scientific discoveries into effective therapy.

Trials for HIV vaccine?

Using a plasmid DNA that encodes protein antigens from HIV

Problems with gene therapy?

Usually due to the vectors used. May cause immune response, have too low of cloning capacity to deliver the gene, or disrupt genes or gene activity

How can viral vectors be used for gene therapy?

Viral vectors are often used to deliver therapeutic DNADNA into host cells.

Viral vectors for gene therapy

Viral vectors carry therapeutic DNA as payload. Virus will infect target cells and successfully deliver the DNA

How are adults tested for SCA? Prenatal?

WBC or cheek Amniocentesis or CVS

How are adults tested genetically? Newborns?

White Blood Cells Heel prick

Why were white blood cells chosen as the targets for the therapeutic gene?

White blood cells, T cells in this case, are key players in the mounting of an immune response, which Ashanti was incapable of developing.

CRISPR-Cas

allows for genome editing by targeting specific sequences in the genome

gene therapy

delivery of therapeutic genes into patient to correct genetic disease conditions cause by faulty genes. Is an example of precision medicine

ex vivo gene therapy

gene is inserted into cells that have been removed and then returned to the body

RNA interference (RNAi)

introduction of double-stranded RNA into a cell to inhibit gene expression

Traditional Gene Therapy

involves addition of a therapeutic gene that coexists with the defective copy.

gene editing

involves removal, correction, or replacement of a defective gene. It can alter one or several bases of a gene or replace the gene entirely.

What are gene-silencing techniques used for gene therapy?

the use of antisense RNA and RNA interference

In vivo gene therapy

therapeutic DNA is introduced directly into the affected cells of the body. The challenge with this is restricting delivery to intended tissues and not all tissues throughout the body.

immunotherapy

use of immune cells, antibodies, or vaccines to treat or prevent disease. Involves genetic engineering of T cells.

What are the two approaches to RNA delivery?

~inject siRNA directly ~Deliver via DNA plasmid vector