Chapter 6: Genetic Strategies in Therapeutics Flashcards
Where do restriction endonucleases cut DNA fragments specifically?
cut DNA specifically at palindrome sequences, yielding restriction fragments of chromosomes
What is the natural source of restriction endonucleases used to cut specific DNA sequences?
bacteria
What is the purpose of restriction endonucleases in bacteria?
act as part of a restriction/modification system that protects that bacterium from infection by DNA viruses
What are palindromes?
double-stranded DNA sequences called palindromes (inverted repeats usually of 4-8 base pairs in length
How can you identify a palindrome?
examine the sequence of only one strand. Then draw a line through the center of the sequence. If the sequence is folded along this line, the bases should pair
What is a vector?
a piece of DNA (plasmid, viral chromosome, yeast chromosome) capable of autonomous replication in a host cell
Which can be used in gene therapy or constructing transgenic animals? genomic or cDNA libraries?
cDNA
What is the reason for cloning EPO and Factor VIII in mammalian cells instead of Ecoli?
because mammalian cells are eukaryotic and therefore have Golgi to perform N glycosylation
Gene transfer requires a delivery vector? What are some common delivery vectors?
retroviruses, adenoviruses, liposomes
What are some advantages of adeno-associated viruses?
having no disease association in humans and limited innate immunity
Describe genome of retroviruses?
ssRNA
Describe genome of adenoviruses?
dsDNA
Describe genome of AAV (Adeno-associated virus)?
ssDNA
What are the names of the 2 strategies for delivering a therapeutic gene (transgene) into an individual?
In vivo
Ex vivo
What is In vivo gene replacement therapy?
involves the direct delivery of a therapeutic gene into a patient’s body
What is ex vivo gene replacement therapy?
involves the genetic manipulation of a patients target cells outside the body
target cells are infected with recombinant virus harboring the therapeutic transgene.The modified target cells, harboring and expressing the therapeutic proteins are then reintroduced into the same patient.
What are some challenges to gene replacement therapy?
- Targeting the therapeutic gene to the appropriate tissues
- Low-level or transient expression of the therapeutic gene
- problems caused by random insertion of the therapeutic gene into host DNA
What is RNAi
refers to downregulation of gene expression through use of small RNA molecules, which interfere with gene expression by either inhibiting translation or causing premature degradation of the genes mRNA
What are the 2 types of small RNA molecules involved in RNA interference?
microRNA (miRNA)
Small interfering RNA (siRNA)
The RNAi pathway has what function in eukaryotes?
plays a central role in defending cells against viruses and transposons
The RNAi process begins when an enzyme cleaves long double stranded RNA into small double stranded RNA fragments. What is the name of this enzyme?
dicer
In RNAi the double stranded siRNA formed from cleavage by the dicer enzyme unwinds into 2 single-stranded RNAs? What is the name of them?
passenger strand (sense strand)
guide strand (antisense strand)
Which siRNA fragment in the process of RNAi degrades initially?
the passenger strand (sense strand)
The RISC associates with which siRNA strand in the process of RNAi?
guide strand (antisense strand)
What does RISC stand for?
RNA-induced silencing complex
The RISC guide strand complex pairs with a complementary sequence in messenger RNA and induces cleavage using what?
argonaute (the catalytic component of the RISC complex
Why is the RNAi effect called knockdown instead of knockout?
because gene expression continues though in greatly reduced extent
compared to “knockout” in which by contrast, gene expression is entirely absent
