[21.2] in vivo gene cloning - the use of vectors

Question 1

what is in-vivo gene amplification?

Answer 1

transferring fragments of DNA to a host cell using a vector

Question 2

what are the 6 key stages involved in in vivo gene cloning?

Answer 2

1. identifying and isolating a specific DNA fragment
2. adding promoter / terminator / marker genes
3. inserting DNA fragment into a vector
4. transforming host
5. identifying successfully transformed hosts
6. growth / cloning of successful hosts

Question 3

what is the importance of sticky ends when DNA is cut by restriction endonucleases?

Answer 3

• if the same RE is used to cut different DNA segments, all the frgments will have ends that are complementary to each other
• single-stranded end of any one fragment can be joined to the single-stranded end of any other fragment

Question 4

what is the role of DNA ligase?

Answer 4

binds the phosphate-sugar backbone of the two sections of DNA once the complementary bases of sticky ends have paired up

Question 5

why do promoter genes have to be added to prepare the DNA fragment for insertion?

Answer 5

• for transcription of any gene to take place, RNA polymerase (which synthesises mRNA) must attach to promoter region of DNA, near a gene
• nucleotide bases of promoter attach to RNA polymerase and transcription factors so being the process of transcription

Question 6

why do terminator genes have to be added to prepare the DNA fragment for insertion?

Answer 6

terminator region of DNA is needed to release RNA polymerase, ending transcription at the appropriate point

Question 7

why do marker genes have to be added to prepare the DNA fragment for insertion?

Answer 7

to identify whether a gene has been taken up by bacterial cells ie. indicates whether transcription has worked

Question 8

what is a vector?

Answer 8

carrying unit used to transport DNA into the host cell

Question 9

what happens when a DNA fragment is inserted into a vector?

Answer 9

1. REs cut part of (plasmid) loop
2. sticky ends opened-up plasmid are complementary to sticky ends of DNA fragment
3. when DNA fragments are mixed with opened-up plasmids, they may become incorporated into them
4. if incorporated, DNA ligase permanently joins them
5. plasmids now have recombinant DNA

Question 10

what is transformation?

Answer 10

when DNA, which has been incorporated into at least some of the plasmids, is reintroduced into bacteria cells

Question 11

why do not all of the transformed bacterial cells have the DNA fragments with the desired gene for the desired protein? (3)

Answer 11

• only a few bacterial cells (1%) take up plasmids when the two are mixed together
• some plasmids close up again without incorporating the DNA fragment
• DNA fragments ends can join up together to form its own plasmid

Question 12

what characteristics might marker genes have which make successful genes easily identifiable?

Answer 12

• resistant to an antibiotic
• make fluorescent protein that is easily seen
• produce an enzyme with visual signal

Question 13

what is the advantage of using fluorescent marker genes rather than antibiotic gene markers?

Answer 13

• with antibiotic-resistance markers, bacterial cells with requored gene are killed, so replica plating is need to obtain cells with gene
• with fluorescent gene markers, bacterial cells are not killed so replica plating is not needed so process is more rapid

Question 14

what are the advantages of in vivo gene cloning?

Answer 14

• useful when we want to introduce a gene into another organism
• lower risk of contamination as contaminant DNA will not be taken up by the plasmid as it is not complementary
• very acurate, DNA copied has few errors
• cuts out specific genes
• produces transformed bacteria that can be used to produce large quantities of gene products

Question 15

describe the basic steps of gene therapy (4)

Answer 15

1. stem cells removed from patients
2. stem cells treated outside of patient’s body
3. modified stem cells returned to patient’s body by vector
4. disease treated based on stem cell’s modification

Question 16

what are the challenges of gene therapy? (9)

Answer 16

1. only works if you deliver a normal gene to a large number of cells (several million) in a tissue, activate it, and make it stay on
2. have to make sure gene isn’t incorporated into wrong cells
3. delivering gene to wrong tissue can cause health issues
4. gene-delivery vectors have to be less likely to trigger an immume response. maintain a germ-free environment?
5. gene can stitch itself into an inappropriate location, disrupting other genes
6. ethical issues + harm caused if complications arise
7. high cost of development makes it unappealing for pharmaceutical companies, so it is less researched
8. very expensive, so some patients may never be able to afford them
9. individualising is expensive eg. compared to bulk drug treatments