Genetic engineering (TK) Flashcards
(22 cards)
What is recombinant DNA?
What are the 4 molecules need to form recombinant DNA?
An artificial stretch of DNA assembled in a precise way
- Restriction endonucleases (Like scissors)
- DNA ligase (like glue)
- Plasmids (like the work bench)
- E.coli (like the factory)
What is a bacteriophage?
A virus that infects bacteria
How does bacteriophage attack work and what do restriction endonucleases do?
Attach to the outside of the bacteria and inject their viral genome into the bacteria. The viral genome is then replicated in the bacteria
Restriction nucleases restrict the infection of bacteriophages by cutting short DNA sequences in the entering bacteriophage genome (cuts the genome up)
Restriction endonucleases cut double stranded DNA sequences
What type of sequences do restriction enzymes recognise and cut, and where in the sequence do they cut?
ALWAYS palindromic sequences
Palindromic = same sequence but backwards on the opposite strand (see summary sheet)
Always cut between the same 2 nucleotides (e.g. between the G and A of both strands) - Hydrolyses the phosphodiester bond between these 2 bases
What prevents EcoRI (and other restriction endonucleases) from cutting the bacterial genome (it’s own genome)?
The bacterial genome is methylated, which prevents the EcoRI from cutting it
What are the 3 types of DNA sequences that are left after being cut with restriction endonucleases?
- 5 prime overhang with sticky ends - most bases are on the 5 prime end
- 3 prime overhang with sticky ends - most bases are on the 3 prime end
- Cut in half with blunt ends8
What does T4 DNA ligase do and what 3 things does it require to do this?
“Sticks” endonuclease strands back together (reforms the phosphodiester bond)
Requires ATP
Requires nearby 5 prime phosphate and 3 prime hydroxyl groups
Requires “sticky” ends - hydrogen bonding between base pairs found only in 5 prime and 3 prime overhangs (more efficient at sticking 5 prime and 3 prime overhangs back together than blunt ends) (Blunt end ligations still occur, just less efficient)
What are the 3 important sites contained in a plasmid?
- Unique cloning sites (restriction sites) - site that is cut to allow recombinant DNA to be inserted
- Drug-resistance gene
- Origin of replication
What is a double digest in plasmids and how is this good?
Inserting DNA using 2 restriction enzymes and 2 specific restriction sites
DNA cannot self-ligate back to the original plasmid, like it can when there is only one type of restriction enzyme
How is bacteria, like E.Coli, used to multiply the recombinant DNA?
(How does the origin of replication and drug resistant gene have a role in this?)
Recombinant DNA is “transformed” into the E.coli
Only the bacteria containing the recombinant DNA plasmid will survive to multiply and produce a visible colony - drug applied, and only recombinant DNA has the drug resistant gene, so only these bacteria will be in the colony
Origin of replication allows the plasmid to be maintained and replicated within the bacteria cell - allows multiple copies of DNA to be produced per cell
How is PCR used to insert the gene of interest when it doesn’t have restriction sites? (4)
PCR primers with adaptors (added restriction sites) are used and bind to the target sequence (gene of interest).
This forms a blunt PCR product with added restriction sites. This is then digested with the specific restriction enzymes to form the sticky ends and is then ligated into the plasmid
What are transgenic animals, how are they formed, and why is this useful to do?
Transgenic animals = genetically modified animals
Formed by microinjecting a zygote with recombinant DNA - zygote is only 1 cell, so changes to the genome at this stage will be inherited by all cell of the organism
Useful for understanding the function of genes and the proteins that they encode
What are the 3 important regions of the transgene to be inserted, what do they contain, and what do they do?
- Ubiquitous transcriptional factor:
Drives expression of downstream gene of interest in ALL tissues at ALL times of development - bent arrow after this region represents TRANSCIPTIONAL initiation (not translational) (see summary sheet for diagram)
Contains a TATA sequence - Polyadenylation signal:
Designates the site of transcriptional termination (where the RNA polymerase will fall off) and initiates the formation of a poly(A) tail
Contains 2 AATAAA signals - Gene of interest e.g. GFP
Give an example of a ubiquitous transgenic animal, and a non-ubiquitous transgenic animal and how they are both formed?
Transgenic mice with GFP (green fluorescence) = ubiquitous, recombinant gene is injected into the zygotes and cultured in a petri dish until they form 2-cell embryos (zygote = only 1 cell). The 2-cell embryos are then surgically transferred to the mother’s fallopian tube
Transgenic cows with medicinal milk = non-ubiquitous, only genes in the mammary gland of cows are modified (see summary sheet for how insert is made)
What is the difference between random transgene integration and genetically engineered embryonic stem cells as methods for transgene integration?
Random transgene integration = integrates into a random chromosome - can result in multiple copies of the transgene, can end up landing on a natural gene in a chromosome and disrupt it
Embryonic stem cells = precise integration into the genome (can control which chromosomes its integrated into) - single copy integration, ideal method to “Knockout” genes
Where are embryonic stem cells derived from and what is good about this?
Blastocysts, which are a later stage embryo than a zygote.
Have lots of cells at this stage, not just one.
Lots of cells means we have lots of attempts
What is a chimera, and how are chimeras made?
What do chimera mice look like, and how do they technically have 2 sets of parents?
What happens when a chimera containing transgenic embryonic stem cell derived eggs/sperm breads with an Agouti?
Chimera = mixture of natural cells and injected modified cell
Made by injecting transgenic embryonic stem cells and a non-transgenic host’s blastocyst
In mice = half white and half brown (half albino, half agouti)
2 sets of parent - non-transgenic parents who made the blastocyst (albino) and the parents that the transgenic embryonic stem cells came from (agouti)
Offspring is agouti, as genetic manipulations in the embryonic stem cells will be passed onto the next generation (genetic manipulation is that the mice are brown)
How is the recombinant DNA inserted into the specific chromosome? (3)
How are these modified embryonic stem cells then cloned, and what can be used to identify the modified genes?
Need to knockout the critical exon (start codon) to manipulate the gene within the genome of the embryonic stem cells.
The exon is broken using restriction enzymes to cut the critical exon and break it
The recombinant DNA is then inserted into the specific chromosome where the critical exon was broken
Cloned by culturing them in a petri dish and adding a drug (to form drug-resistant colonies)
Southern blotting can be used to identify the correctly targeted clones with the modified gene in them
How can homologous Fgf5 wild-type chromosomes occur (where both don’t have the recombinant DNA)?
What is observed in the southern blot with heterozygous vs homozygous ?
What can the crossing over of 2 heterozygous Fgf5 modified genes form, and what would the mouse formed look like?
Recombination of the 2 chromosomes (very rare) (see summary sheet diagram) - results in offspring being homologous for the Fgf5 wildtype (both are normal, without any recombinant genes) (Fgf5 +/+)
Heterozygous = 2 spots, one at 3.5kb and another at 5.3kb (Fgf5 +/-)
Homozygous = 1 spot at 3.5kb (both only have ex1 instead of the insert) (Fgf5 +/+)
Can result in a knockout mouse (mouse without any Fgf5 normal gene, only the recombinant version), forms an extra hairy mouse (Fgf5 -/-)
What are 2 other examples of knockout genes in mice, and what do they result in (not Fgf5)
Leptin knockout = extra big mice
Myostatin knockout = extra muscly mice
What do designer nucleases do?
How are designer nucleases, like CRISPR/Cas9 different to restriction nucleases?
Cut DNA (in embryos)
Different as uses a single RNA guide to find and cut the specific sequences - adds an RNA sequence that is complementary to the genomic region of interest (Can be specific to multiple sequences, as just need to change the guideRNA to change the specificity - unlike restriction nucleases that are only specific to one DNA sequence) (see diagram on summary sheet)
Why is gene targeting using CRISPR/Cas9 so useful?
What are 2 examples of when CRISPR/Cas9 is used and what can it cause to happen?
No longer need to use embryonic stem cells, can make cuts within the target gene of the zygote
Fgf5 - using Fgf5 specific CRISPR/Cas9 - can cause knockout (both chromosomes having the modified gene) (see diagram on summary sheet)
Tyr - using Tyr specific CRISPR/Cas9 - can cause knockout, albinoism in mice pups
