Chapter 6: Genetic Strategies in Therapeutics

Question 1

Where do restriction endonucleases cut DNA fragments specifically?

Answer 1

cut DNA specifically at palindrome sequences, yielding restriction fragments of chromosomes

Question 2

What is the natural source of restriction endonucleases used to cut specific DNA sequences?

Answer 2

bacteria

Question 3

What is the purpose of restriction endonucleases in bacteria?

Answer 3

act as part of a restriction/modification system that protects that bacterium from infection by DNA viruses

Question 4

What are palindromes?

Answer 4

double-stranded DNA sequences called palindromes (inverted repeats usually of 4-8 base pairs in length

Question 5

How can you identify a palindrome?

Answer 5

examine the sequence of only one strand. Then draw a line through the center of the sequence. I the sequence is folded along this line, the bases should pair

Question 6

What is a vector?

Answer 6

a piece of DNA (plasmid, viral chromosome, yeast chromosome) capable of autonomous replication in a host cell

Question 7

Which can be used in gene therapy or constructing transgenic animals? genomic or cDNA libraries?

Answer 7

cDNA

Question 8

What is the reason for cloning EPO and Factor VIII in mammalian cells instead of Ecoli?

Answer 8

because mammalian cells are eukaryotic and therefore have Golgi to perform N glycosylation

Question 9

Gene transfer requires a delivery vector? What are some common delivery vectors?

Answer 9

retroviruses, adenoviruses, liposomes

Question 10

What are some advantages of adeno-associated viruses?

Answer 10

having no disease association in humans and limited innate immunity

Question 11

Describe genome of retroviruses?

Answer 11

ssRNA

Question 12

Describe genome of adenoviruses?

Answer 12

dsDNA

Question 13

Describe genome of AAV (Adeno-associated virus)?

Answer 13

ssDNA

Question 14

What are the names of the 2 strategies for delivering a therapeutic gene (transgene) into an individual?

Answer 14

In vivo
Ex vivo

Question 15

What is In vivo gene replacement therapy?

Answer 15

involves the direct delivery of a therapeutic gene into a patient’s body

Question 16

What is ex vivo gene replacement therapy?

Answer 16

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.

Question 17

What are some challenges to gene replacement therapy?

Answer 17

1. Targeting the therapeutic gene to the appropriate tissues
2. Low-level or transient expression of the therapeutic gene
3. problems caused by random insertion of the therapeutic gene into host DNA

Question 18

What is RNAi

Answer 18

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

Question 19

What are the 2 types of small RNA molecules involved in RNA interference?

Answer 19

microRNA (miRNA)
Small interfering RNA (siRNA)

Question 20

The RNAi pathway has what function in eukaryotes?

Answer 20

plays a central role in defending cells against viruses and transposons

Question 21

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?

Answer 21

dicer

Question 22

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?

Answer 22

passenger strand (sense strand)
guide strand (antisense strand)

Question 23

Which siRNA fragment in the process of RNAi degrades initially?

Answer 23

the passenger strand (sense strand)

Question 24

The RISC associates with which siRNA strand in the process of RNAi?

Answer 24

guide strand (antisense strand)

Question 25

What does RISC stand for?

Answer 25

RNA-induced silencing complex

Question 26

The RISC/ guide strand complex pairs with a complementary sequence in messenger RNA and induces cleavage using what?

Answer 26

argonaute (the catalytic ocmponent of the RISC complex

Question 27

Why is the RNAi effect called knockdown instead of knockout?

Answer 27

because gene expression continues though in greatly reduced extent

compared to “knockout” in which by contrast, gene expression is entirely absent