M3 L18: Recombinant DNA Technology

Question 1

what are restriction enzymes? original function?

Answer 1

break DNA at specific sequences

protect bacteria from invading nucleic acids

Question 2

how do bacteria prevent self cleavage from restriction enzymes

Answer 2

own DNA is methylated

Question 3

what are sticky ends

Answer 3

result of restriction enzymes making staggered cuts that yield complementary overghangs

Question 4

what is molecular cloning

Answer 4

digest vector and DNA of interest w/ same restriction enzyme –> mix so sticky ends anneal and add DNA ligase (2 types recombinant plasmids and poss. nonrecombinant) –> put vector in organism –> vector/DNA replicates when organism replicates (make DNA clones)

Question 5

whats a vector

Answer 5

carrier molecule that holds DNA and allows it to be replicated in a biological system

Question 6

what is directional cloning? what’s it used for?

Answer 6

using 2 dif restriction enzymes –> non complementary sticky ends so vector can’t re-anneal to itself

used for expression vectors where it matters which side of the DNA of interest is next to the promoter and termination seqs

Question 7

4 properties of plasmids used in recombinant DNA technology

Answer 7

1) don’t have seqs that will recombine with host (like F plasmid does)

2) have a selectable marker to distinguish from cells without the plasmid (like antibiotic resistance)

3) have origin of replication –> fast replication in host

4) have a multiple cloning site (MCS): can be cut by many dif restriction enzymes - cannot be cut anywhere else

Question 8

how to replicate your plasmid/vector

Answer 8

put it in E. coli (transormation - bacterial cell takes up DNA from environment)

mix E. coli and plasmid, shock to make competent cells, use [plasmid] where each cell takes one

Question 9

what’s a pUC-based plasmid? what do the results mean?

Answer 9

pUC-based plasmid: MCS is in the lacZ gene; plate bac with antibiotic and X-gal

Any colony present: transformed with vector (acquired antibiotic resistance)

Blue: X-gal was digested; means B-gal present; means lacZ uninterrupted and colony was not transformed with recombinant plasmid

white: X-gal not digested; means B-gal not present; means lacZ interrupted and colony was transformed with recombinant plasmid

Question 10

What are BACs and YACs? when are they used?

Answer 10

bacterial and yeast artificial chromosomes

accommodate larger DNA inserts than plasmids

BACs: 100-200 bp, origin of replication from F factor –> 1-2 copies per cell; have a marker and MCS

YACs: 1000 kb

Question 11

what are DNA libraries? what are the two types and what do they reflect?

Answer 11

DNA library: collection of cloned frags of DNA from nucleic acids from a single organism

genomic libraries (gDNA): from genomic DNA, including introns and exons –> seq a genome; abundance reflects copy number

complementary libraries (cDNA): reverse transcribed mRNA, only includes exons; can vary btwn dif tissues in same organism –> analyze patterns of expression; abundance reflects expression level

Question 12

what is a potential complication for artificial synthesis of DNA molecules

Answer 12

codon optimization/codon bias: species vary in their relative abundance of different tRNAs –> original species and vector species might have different preferred codons –> slow translation, misfolding, low expression

Question 13

how to overcome codon bias

Answer 13

codon optimization: make a protein sequence that’s synonymous to the original species using preferred codons of the vecor/expressing species

Question 14

what are transgenes/transgenic organisms

Answer 14

transgene: gene that’s introduced into another organism

transgenic organism: organism that has a transgene (gene from another organism)

Question 15

what are heterologous transgenes

Answer 15

transgenes from different species

Question 16

2 major challenges for making transgenic organisms

Answer 16

1) need to introduce DNA molecule in a way that it integrates into host genome

2) need to provide appropriate regulatory sequences

Question 17

what allows for the making of transgenic organisms/expression

Answer 17

universality of genetic code

Question 18

how to express heterologous genes in bacteria and fungi (usually human genes)

Answer 18

transformation of a recombinant plasmid

use expression vector with a promoter and shine dalgarno seq upstream MCS and transcription terminator downstream MCS

need to remove introns (use cDNA transgenes reverse transcribed from euk mRNA)

Question 19

when to use yeast over bacteria/fungi

Answer 19

if trying to express euk transgene that requires post translational modifications

Question 20

application of heterologous gene expression for insulin

Answer 20

insulin previously harvested from pig/cow pancreas (not pure –> risk allergic reaction)

make insulin by inserting gene into E. coli –> pure

Question 21

application of heterologous trnasgenes for HGH

Answer 21

previously harvested from cadaver pituitary gland (not pure –> risk creutzfelt-jacob disease)

insert HGH gene into E. coli –> pure

Question 22

what is agrobacterium transformans

Answer 22

bacteria

wild strains carry Ti plasmid (tumor inducing) –> cause grown gall disease

Question 23

what is T-DNA

Answer 23

part of the Ti plasmid that gets transferred to the nucleus of the plant –> can recombine with plant genome at a random location

has regulatory seqs that allow for efficient expression in plant but not bacteria

Question 24

T-DNA encodes genes that can do what 2 things

Answer 24

1) cause plant cells to divide uncontrollably –> also replicates the bacteria

2) cause plant to make lots of opines (carbon/nitrogen source for bacteria but not plant)

Question 25

What genes are on the Ti plasmid

Answer 25

T-DNA genes

genes for transferring DNA to the host plant cell (homologous to F plasmid)

Question 26

how to make a transgenic plant

Answer 26

disarm Ti-plasmid (remove opine and tumor genes from T-DNA, replace w/ gene of interest and a selectable marker like antibiotic or herbicide resistance)

insert modified Ti plasmid into totipotent plant cell genome –> grow into adult plant

Question 27

what are the two plasmids used in the binary approach for making transgenic plants

Answer 27

1) disarmed Ti plasmid (for transfer machinery)

2) transformation vector with gene of interest and selectable marker and T-strand border sequences to make sure other genes are transferred

Question 28

what’s the most common desirable trait when making transgenic plants? pros and cons?

Answer 28

herbicide resistance (to kill weeds)

pro: round up ready corn saves on tilling

con: using more herbicides like round-up may increase cancer risk

Question 29

other desirable trait for transgenic plants? how to achieve?

Answer 29

resistance to insects

make plants that express Bt toxins from a bacteria –> tocix to certain insects but not humans

Question 30

what’s challenging about making transgenic animals

Answer 30

homologous recombination (recombination at a specific site) is less common than illegitimate recombination (at a random site)

most animal cells are not totipotent –> can’t just insert transgene into any cell and grow it into an adult (need to modify eggs, embryos, or cells that give rise to gametes)

Question 31

why/how to prevent 2nd meitotic division in transgenic salmon

Answer 31

how: pressure treat eggs

why: make triploid offspring so indiv w/ transgene cannot mate with wild pop

Question 32

how are the phenotypes variable if you insert the same growth hormone transgene into salmon

Answer 32

illegitimate recombination –> growth hormone gene inserted in dif places –> dif effect if in hetero vs euchrom region

Question 33

how to knock out genes in mice

Answer 33

replace central region of CFTR mouse gene with positive selectable marker (drug resistance) –> negative marker downstream (drug sensitivity)

only cells with homologous recomb will have positive marker and will survive –> inject in mouse blastocyst w/ dif genotype (ex coat color)

grow mosaic –> cross to parent –> if offspring have recomb phen, then recombinant genotype in germline

breed het F1s –> 25% F2s homo recombinant (do not have neg marker gene) –> confirm genotype w/ PCR

Question 34

what’s gene therapy and the two types

Answer 34

use genes to cure/alleviate disease symptoms

somatic gene therapy: extract cells, fix the problem, re-introduce cells or deliver gene to affected tissue; not heritable

germline gene therapy: not in humans bc of possible off target effects of crispr - use crispr-cas9 to edit gene in cells that give rise to germline

Question 35

unique challenges of somatic gene therapy

Answer 35

only some tissue types can be removed and introduced (blood but not lungs)

Question 36

what are embryonic stem cells? why are they ideal for somatic gene therapy?

Answer 36

totipotent cells present in embryo

can be edited by crispr-cas9 and induced to develop into certain type of tissue to fix the problem

Question 37

how to make equivalent of embryonic stem cells

Answer 37

isolate fibroblasts –> reprogram by expressing yamakana factors (use disarmed viral vectors to deliver yamanaka transgenes into host genome) –> alters chromatin to ESC-like state

Question 38

3 potential issues expressing yamanaka factors

Answer 38

1) might disrupt other genes if integrated in wrong location

2) continued expression –> cancer

3) need to stop expressiona t some point: can flox yamanaka factors for later removal via cre-recombinase)

Question 39

mechanism to cure sickle cell in mice

Answer 39

isolate tail fibroblasts –> induce expression of yamanaka factors to un-differentiate fibroblasts into ESC-like state –> use crispr-cas9 to edit sickle cell gene –> re-differentiate cells to hematopoietic stem cells –> put back in irradiated mice (radiation knocks out original mutated HPSCs)

Question 40

3 downsides of using ESCs to cure genetic diseases

Answer 40

1) off target effects of transgene insertion

2) yamanaka factor expression may cause cancer

3) not applicable for cells that can’t be removed and replaced

Question 41

how do the 2 gene therapy approaches to cure sickle cell in humans work

Answer 41

both use crispr-cas9 to edits HPSCs

Question 42

You are cloning a human gene responsible for sequestration of heavy metals in the cytoplasm. You use a pUC-based plasmid. Following construction of your recombinant vector, your transformation only yielded blue colonies. What does this indicate?

Answer 42

Blue colonies means B-galactosidase was present and able to break down X-gal. Since there is functional B-gal, the LacZ gene was not interrupted. This means the DNA sequence was not inserted in the plasmid.

Question 43

Compare and contrast the F plasmid with the Ti plasmid.

Answer 43

Similar: encode machinery for gene transfer; similar mechanisms of conjugation with exporter proteins, relaxosome, relaxase; allow for one-way transfer of genetic material between cells

Different: Whole F plasmid is transferred and both cells have a copy afterward; double stranded. Only part of the Ti plasmid (T-DNA) gets transferred to the plant cells and it’s single stranded.

Question 44

Explain how you would use a two-plasmid approach to introduce a Bt toxin gene into corn. What genes would be on each plasmid? How would you isolate transformed cells?

Answer 44

Use a Ti plasmid with the T-region removed (disarmed) to transfer genetic material to another bacterial cell via conjugation. Another plasmid acts as a transformation vector with Bt toxin gene, herbicide resistance gene as a marker, and T-strand border sequences. Put both plasmids into corn cells. Isolate transformed cells by plating with herbicide.