Tissue Engineering Quiz 5

Gene Therapy

technique that uses genes to treat or prevent disease by altering a patient's cells instead of relying on drugs or surgery

Gene Replacement

replacing faulty genes with healthy ones

Gene Silencing

turning off genes that work improperly

Gene Addition

adding new genes to fight disease.

Gene Editing

permanently changing genes in the genome

What is the role of gene delivery in tissue engineering (TE)?

enables genetic modification to produce proteins, protect tissues, and stimulate tissue growth or differentiation

Ex Vivo Gene Therapy

genes are introduced into cells outside the body, and the modified cells are then transplanted back into the patient

In Vivo Gene Therapy

genes are delivered directly into the patient's body, targeting cells in their natural environment.

How does hematopoietic stem cell gene therapy work?

Genes introduced into patient's blood-forming stem cells outside the body, and modified cells are transplanted back to treat genetic or blood-related disorders

What are some issues related to gene transfer?

ensuring efficient delivery, avoiding immune reactions, maintaining long-term gene expression, and minimizing off-target effects

Why use gene therapy?

uses the body's own cells to produce therapeutic proteins, making it more efficient, cost-effective, and long-lasting with various therapeutic applications

What happened to Jesse Gelsinger in 1999?

died from multiple organ failure four days after participating in a gene therapy trial for OTCD, likely due to an immune response to the adenovirus carrier

What did the NIH panel say about gene therapy?

great potential but still faces major challenges, such as issues with gene transfer vectors and understanding their interaction with the body

What are the challenges in gene therapy?

low gene transfer success, lack of quantitative data, insufficient controls, and poorly defined endpoints in gene therapy trials

Non-Viral Vectors

Methods like plasmids or liposomes used to deliver genes without using viruses

Example of Non-Viral Vectors

Plasmid, Plasmid+Liposome

Viral Vectors

Modified viruses used to deliver genes into cells

Example of Viral Vectors

Adenovirus, Adeno-Associated Virus, Lentivirus, Retrovirus

Compared to non-viral delivery, viral delivery is...

more effective, more likely to generate stable transfections, less safe, more expensive, and takes more time

Methods of non-viral gene delivery

mechanical (microinjection, gene gun), electrical (electroporation), and chemical (DEAE-dextran, calcium phosphate, and lipids/proteins/polymers)

Hurdle with direct injection into the nucleus

it's impractical to inject DNA into many cells

Hurdle with electroporation

need to be very careful not to damage the cells since your using electrical pulses to temporarily open the cell membrane, allowing DNA to enter

DEAE-dextran

positively charged polymer that helps DNA enter cells by binding to the negatively charged cell membrane, useful for transient gene delivery but not for stable integration

Cationic Polymers

Contain positive charged groups formation of polyplexes with DNA. Relatively inexpensive. Ability to incorporate ligands

Liposomes

synthetic carriers that bind to (-) DNA and cell membranes, and can be improved with additives to better stabilize and work under various conditions

LIPOFECTAMINE PLUS transfection procedure

mixing DNA with Lipofectamine Plus reagent to form complexes that help the DNA enter cells for gene expression

Gene Gun

Gold or tungsten particles coated with DNA are loaded into a plastic bullet causing the DNA-coated particles to hit the target tissue

Electroporation

electric pulse temporarily creates pores in the cell membrane, allowing the DNA to enter the cell before the membrane reseals

Concerns with Gene Therapy

short term nature, immune response, viral vector problems, multigene disorders

Integrated Transgenes

transgenes that are permanently added to the host genome and passed to future cells

Non-Integrated Transgenes

transgenes that stay outside the genome and are temporary

Why can retroviral vectors infect only dividing cells?

Retroviral vectors need the nuclear membrane to break down during cell division to enter and integrate into the chromosome

Adenoviruses

Viruses with double-stranded DNA that can infect both dividing and non-dividing cells; serotypes 2 and 5 are often used as vectors

Problems with Adenoviral Vectors

They cannot integrate into the host genome and gene expression is short lived

Adeno-Associated Virus (AAV)

ideal for gene therapy since it doesn't cause inflammation, avoids triggering antibodies, can enter non-dividing cells, and integrates into a specific site in the genome

Biotechnology

use of microbes to make a protein product

Recombinant DNA Technology

insertion or modification of genes to produce desired proteins

Genetic engineering

manipulation of genes/insert DNA into cells

Gene cloning

isolating genes from one organism, manipulating purified DNA in vitro, and transferring to another organism

Why is genetic engineering important?

producing proteins (like insulin), amplifying specific genes, and studying gene function and regulation

Steps of plasmid cloning

cut the DNA sample and plasmid with enzymes -> join them together -> introduce them into cells -> grow the cells on plates and select for antibiotic resistance

Explain the transformation step in plasmid cloning

process of transferring exogenous DNA into cells

What are some ways to do the transformation step in plasmid cloning?

electroporation or CaCl2 and heat shock

What is transformation?

when a cell takes up and expresses a new piece of DNA to change the organism's traits

What is a gene?

piece of DNA which provides the instructions for making
(coding for) a particular protein

Bacterial Transformation

Transformation is the process by which foreign DNA is introduced into a cell

pGLO Plasmid

tool used in experiments that makes bacteria glow green under UV light and gives them resistance to antibiotics like ampicillin

Green Fluorescent Protein (GFP)

absorbs light at one wavelength and emits it at a longer wavelength (green) and is used to tag cells to observe under a fluorescence microscope.

What do blue colonies indicate on the agar plate?

Ampicillin-resistant and contain pVector with a functional LacZ alpha gene

What do white colonies indicate on the agar plate?

Ampicillin-resistant and contain pInsert, but they do not produce the LacZ alpha gene

DNA recombination

DNA fragment to be cloned is inserted into a vector

Transformation

recombinant DNA enters into the host cell and proliferates

Selective amplification

specific antibiotic is added to kill E. coli without any protection

Cloning vectors

DNA molecules that are used to "transport" cloned/target sequences between biological hosts and the test tube

Properties of all cloning vectors

replicate independently, have a selection marker, contain unique restriction sites for cloning, and have minimal non-essential DNA to optimize the process

The four major types of vectors are

plasmids, bacteriophages/viruses, cosmids, and artificial chromosomes

What are plasmids?

small, circular pieces of DNA found in bacterial cells that can carry genes allowing bacteria to produce proteins they normally wouldn't

What can plasmids cause cells to do?

produce specific proteins

What are plasmid vectors?

small, circular pieces of DNA used to carry and transfer genes into cells

What are selective markers?

genes that help identify or select cells that have taken up a plasmid, often by making them resistant to antibiotics

What is the origin of replication?

DNA sequence where the replication of DNA begins, allowing the plasmid to replicate in the host cell

What is multiple cloning site?

region in a plasmid that contains several unique restriction enzyme sites, making it easier to insert foreign DNA

How is a vector chosen?

based on size and type of DNA to be cloned

Limitations of plasmids

can only carry small DNA, are hard to transform, require more energy to replicate, and may not pass to all daughter cells.

What is bacteriophage lambda?

virus that infects E. coli, injecting its DNA, which circularizes and starts its life cycle in the host

What are cosmid vectors?

plasmids with lambda cos sites, allowing them to carry larger DNA fragments and be packaged into phage particles for delivery

What are Yeast Artificial Chromosomes?

synthetic DNA molecules that are designed to carry large DNA fragments, making them useful for cloning large genes or studying complex genomes

What are Restriction Endonucleases?

bacterial enzymes that cut double-stranded DNA at specific sequences, creating fragments. They always cut the same sequence in the same way, regardless of the DNA molecule

A piece of DNA can be inserted into a plasmid if...

circular plasmid and the source of DNA have recognition sites for the same restriction enzyme

"sticky ends"

overhangs that can pair and join with other sticky ends using enzymes like DNA ligase, enabling recombination

"blunt" ends

straight cuts without overhangs, making recombination harder

What does a reporter gene do?

produces a measurable signal, indicating if the cell is expressing the protein of interest

What is a virus?

tiny bundle of genetic material coat, or capsid, which is made up of bits of protein called capsomeres

What is the origin of viruses?

plasmids, which are small DNA fragments that replicate but can't form coats or move between cells

What is the origin of plasmids?

they replicate themselves

What do viruses do in nature?

reproduce by taking over a host cell's machinery to make copies of themselves

How do viruses get their genetic material into a host cell?

either trick the cell into pulling them in, fuse their coat with the cell membrane, or inject their genes directly into the cell

What types of proteins do viruses make using the host machinery to multiply and spread?

Gag, Pol, Env

What must RNA include for proper gene regulation?

long terminal repeats (LTRs) and cis-acting elements, which are DNA elements that regulate transcription

What is the function of the long terminal repeat (LTR)?

the control center for gene expression

What are packaging cell lines used for in virus production?

provide the machinery to produce retroviruses but lack RNA and regulatory elements, allowing you to create retroviral vectors by adding the desired RNA

What components do packaging cell lines produce?

gag, pol, and env proteins required for retrovirus assembly

What are induced pluripotent stem (iPS) cells?

adult cells reprogrammed to behave like stem cells

How are iPS cells generated?

introducing specific genes or proteins that reset the adult cells to an undifferentiated state

What does over-expression of Olig1 do?

promotes the formation of oligodendrocytes

What are the advantages of retroviral vectors?

provide permanent gene expression and can be used with pseudotyped viruses

What are the disadvantages of retroviral vectors?

only infect dividing cells and may cause insertional mutagenesis, leading to tumor formation by activating oncogenes or inhibiting tumor suppressor genes

What are the limitations of retrovirus vectors?

can't infect non-dividing cells, have trouble with long-term gene expression, and can randomly integrate into the host DNA

What are lentiviral vectors?

can infect non-dividing cells like hepatocytes and neurons

How are lentiviral vectors made safer for use in gene therapy?

deleting accessory genes, providing a non-retrovirus envelope (like VSV), and using lentiviruses from non-human pathogens like SIV, FIV, and EIAV

What are herpesvirus vectors?

based on herpes simplex virus 1, which causes mild disease in humans and poses no significant risk

What is the target and type of herpesvirus vectors?

neurotropic, meaning they target the nervous system, and are typically replication-defective amplicon particles

What are Adeno-associated virus (AAV) vectors?

non-pathogenic, non-enveloped single-stranded DNA viruses that require a helper virus for replication

What are the advantages of AAV vectors?

integration and persistent gene expression, do not cause insertional mutagenesis, can infect both dividing and non-dividing cells, and are considered safe

What are the disadvantages of AAV vectors?

size limitation, low virus titer, and produce low levels of gene expression

Gene knockout

technique for selectively inactivating a gene by replacing it with a mutant allele in an otherwise normal organism

What is microinjection?

gene is injected into the male pronuclei to create a transgenic animal

How does microinjection work in creating a transgenic animal?

gene is injected directly into the male pronuclei of a fertilized egg, leading to the creation of a transgenic animal

What are the advantages of knock-out technology?

specific loss of a gene in mice, helping study gene function, mimic human diseases, and test treatments

What are the drawbacks of transgenic animals?

randomly integrated DNA, require in vitro fertilization, and may have multiple copies of the inserted gene