Biotechnology 2 - Gene therapy and microarray technology

Question 1

What is gene therapy?

Answer 1

Technique for correcting detective genes responsible for disease development

Question 2

How does gene therapy work?

Answer 2

1. Deliver the therapeutic gene to the target cell
2. Gene translocates into the nucleus
3. Generation of function protein product restores the target cell to normal state

Question 3

Types of gene therapy

Answer 3

1. normal gene inserted into non-specific location to replace a non-functional gene
2. Abnormal gene swapped for a normal gene or made inactive through homologous recombination

Question 4

Delivery of genes/DNA by…

Answer 4

1. transfection and

2. infection

Question 5

What is tranfection?

Answer 5

introducing nucleic acids into cells by non-vial methods

- chemical or physical reagents

Question 6

What is transfection via chemical reagent?

Answer 6

• Liposomes (created by lipid with overall net + charge at physiological pH)
  The cationic portion associates with the negatively charged nucleic acids
  Liposome + nucleic acid complex
  Complex taken up via endocytosis
  Complex transported to nucleus

Question 7

What is transfection via physical methods?

Answer 7

• Direct microinjection (very laborious but efficienct)
• Electroporation (expose cells to electrical impulse to form pores on membrane)
• Biolistic particle delivery (relies on HIGH VELOCITY delivery of nucleic acids to recipient (high speed like gut)

Question 8

Limitation of transfection

Answer 8

Inefficent process (low success rate),
very laboriousness
better gene delivery by tablet/aerosols...

Question 9

Gene delivery by infection

Answer 9

by viruses infection,
can almost be 100% efficient in some cases
By vectors (adenovirus, retrovirus, Herpes simplex virus)
Virus is taken up by vesicles very easily
gene is transported to the nucleus when vesicle degrades

Question 10

Direct gene delivery?

Answer 10

only done by infection
transfect virus with therapeutic gene
By injection

Question 11

Cell-based delivery?

Answer 11

1. we have to take cells out of the patient
2. Transfectic therapeutic gene by infection./transfection
3. Culture it in pestri dish

Question 12

What is the most common genetherapy

Answer 12

Cancer disease, monogenic disorder, Infectious disease

Question 13

Gene therapy success in..

Answer 13

In pt who were born wth blindess.

Glybera for the treatment of lipoprotein lipase deficiency (person lacks protein needed to breakdown fat)

Question 14

Limitation of gene therapy

Answer 14

Short lived (multiple rounds may be necessary)
Immune response –> reduce effectiveness
Problem with vectors (can cause toxicity/immune/inflammatory response)
Multigene disorders

Question 15

What does DNA microarray do?

Answer 15

to study gene expression in high throughput (fast and a lot!) manner,
can be used to measure changes in expression levels in tissue samples

Question 16

What is DNA microarray?

Answer 16

Small orderly arrangement of DNA fragments representing the genes of an organism
(sequences from 1000s of different genes)

Question 17

what are in the spots in DNA microarray?

Answer 17

DNA, cDNA, or oligonucleotides

Question 18

How to use microarray?

Answer 18

Slide it onto which thousands of DNA fragments have been arranged in a grid,
each DNA spot corresponds to a gene (to look at expression level)

Question 19

DNA microarray METHOD

Answer 19

1. Prepare DNA chip using chosen target DNA
2. isolate mRNA from tissue sample in disease/normal
3. Prepare cDNA sample for hybridisation to the microarray chip
4. Incubate hybridisation mixture
5. cDNA sample will hybridize and bind to the DNA chip
6. Detect bound cDNA using laser technology
7. Analyse data using computer methods called bioinformatics

Question 20

How to prepare cDNA samples for hybridisation?

Answer 20

Reverse transcribe your mRNA to make cDNA,

label the samples with fluorescent dyes

Question 21

How does cDNA sample hybridize?

Answer 21

Using ‘base-pairing’
mRNA molecule hybridize specifically to the DNA template from which it originated
When two complementary sequences find each other (target DNA on slide and mobile cDNA), they lock together and hybridize

Question 22

What can microarrays be used for?

Answer 22

measure the changes in gene expression levels,
used for diagnosis, tx, drug development (identify receptors that are overexpressed in some cancers), clinical trials

Detect mutations or polymorphisms in gene sequence (ca assess SNPs)
used for determination of CYP profile

Question 23

Advantage of microarray

Answer 23

Can study many genes at the same time
very fast
very easy to use, non toxic, convenient
DNA sequence information is not needed (we can use genes that are only partially known)

Question 24

Disadvantage of microarray

Answer 24

Requires robots (expensive),
hybridisation with similar genes is possible --> false positives
analysis requires specialised databases