Recombinant DNA techniques

Question 1

3 steps of natural homologous recombination

Answer 1

1. Restriction endonuclease cuts double stranded maternal and paternal DNA making 2 cuts (one in each strand) 2. Strand invasion assisted by base pairing 3. DNA segments joined by ligase

Question 2

Recombinant DNA (rDNA)

Answer 2

Sequences generated from multiple sources, creating DNA that is not normally found in biological organisms. Restriction nucleases cut DNA from 2 sources and make sticky ends which H-bond with each other and then bonded with ligase.

Question 3

Vector

Answer 3

DNA molecule in which the DNA template for the recombinant protein can be cloned

Question 4

3 requirements for prokaryotic vectors (bacterial plasmids)

Answer 4

1. Plasmid replicates itself inside bacterial host as the bacteria replicates 2. Each copy of the plasmid encodes for an antibiotic resistance gene so only bacteria with inserted gene grows 3. Specific nucleotide sequence recognized by restriction endonuclease for recombination

Question 5

Recombinant protein

Answer 5

Protein produced by bacteria that has recombinant DNA inserted in it

Question 6

3 other types of vectors

Answer 6

1. Bacteriophages 2. Retroviruses 3. Bacterial or yeast artificial chromosomes

Question 7

3 advantages of using bacterial systems

Answer 7

1. Easy to culture 2. Grow fast 3. Produce high yields of recombinant protein

Question 8

2 issues with bacterial vectors

Answer 8

1. Multi-domain eukaryotic proteins expressed in bacteria often non-functional because the cells are not equipped to accomplish post-translational modifications/molecular folding 2. Many proteins become insoluble as inclusion bodies which are difficult to recover

Question 9

3 Issues with mammalian in vivo expression

Answer 9

1. Yield is low 2. Cost is high 3. Culturing is time-consuming Alternative? Insect cells

Question 10

Mammalian in vivo expression advantage

Answer 10

Usually produce functional protein

Question 11

Gene therapy

Answer 11

Normal gene inserted into genome to replace abnormal. Target cells are infected with the viral vector which unloads genetic material containing therapeutic gene into target cell. Generation of functional protein product from therapeutic gene restores target cell to normal state.

Question 12

Viral vector in gene therapy

Answer 12

Contains DNA encoding therapeutic product

Question 13

Retroviruses

Answer 13

Can create double stranded DNA copies of their RNA genomes which can be integrated into chromosomes of host cells

Question 14

Adenoviruses

Answer 14

Have double stranded genomes that cause respiratory, intestinal, and eye infections in humans (like common cold virus)

Question 15

Adeno-associated viruses

Answer 15

Single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19

Question 16

Herpes simplex viruses

Answer 16

Double stranded DNA viruses that can infect neurons (herpes simplex virus 1 causes cold sores)

Question 17

2 nonviral gene delivery options

Answer 17

1. Direct introduction of therapeutic DNA into target cells, requires large amounts of DNA and limited in application 2. Creation of artificial lipid sphere with aqueous core. This liposome carries therapeutic DNA and able to pass DNA through target cell’s membrane

Question 18

4 problems with gene therapy

Answer 18

1. Short-lived nature of therapy (need multiple rounds) 2. Immune response (may be attacked, especially in repeat treatments) 3. Problems with viral vectors (possible toxicity, immune/inflammatory responses, virus might become activated) 4. Multigene disorders (very hard to treat with this method)

Question 19

Small interfering RNA (siRNA)

Answer 19

AKA silencing RNA. Double stranded RNA with 2nt 3’ overhangs on either end due to Dicer processing which can interfere with expression of a specific gene.

Question 20

Knockdown by siRNA

Answer 20

siRNA exogenously induced into cells by silencer expression plasmid. Target mRNA for degredation. Varying levels of effectiveness in animals.

Question 21

Transgenic mouse

Answer 21

Carries foreign gene that has been deliberately inserted into its genome

Question 22

3 reasons to use mice as models

Answer 22

1. Physiologically similar to humans 2. Large reservoir of potential models generated 3. Study disease on uniform genetic background

Question 23

Knockout mutation

Answer 23

Replacement of gene segment by homologous recombination results in non-functioning or “null” allele

Question 24

Knock-in mutation

Answer 24

Mutation is a point mutation that results in partially functional or non-functional allele

Question 25

Reporter constructs

Answer 25

Promoter for reported gene that is linked to the the inserted gene which can be assayed

Question 26

Generation of transgenic mice--in vitro recombination steps

Answer 26

1. Isolate gene to be knocked out and made nonfunctional 2. Visible marker and genes to confer resistance to antibiotics added 3. Embryonic stem cells isolated from blastocysts and grown in vitro 4. DNA transferred across membrane by electroporation where some ESCs will incorporate sequence into genome via homologous recombination 5. Use marker to isolate ESCs with sequence from those without

Question 27

Generation of transgenic mice--in vivo breeding steps

Answer 27

1. ESCs with sequence inserted into new mouse blastocyst and implanted in pseudopregnant mouse 2. Newborns will be chimeras (have stem cells of 2 types) 3. Chimeras crossed with wild type 4. Offspring inbred to produce mice with no functional copy of gene (homozygous)