Genetic Engineering Flashcards
Definition
a way of making specific changes to the DNA of a cell or organism. An enzyme cuts the DNA at a specific sequence, and when this is repaired by the cell a change or ‘edit’ is made to the sequence
Gene editing
What does RNAi result in?
Cleavage of target mRNA by the RNA-induced silencing complex (RISC)
True or False:
Cre expression can be activated at a specific time by a specific signaling molecules
True
Definition
a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system
PAM site
Define
Transcription activator-like effectors (TALE)
proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species
What are the advantages of morpholinos?
Quick
Cheap
Definition
the introduction of a transgenes that has the potential to change the phenotype of an organism
Transgenesis
Out of adenovirus and lentivirus vectors, which integrate into the genome?
Lentivirus
Define
Knockins
a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus
Define
Homology-directed repair
a mechanism in cells to repair double strand DNA lesions. The most common form of HDR is homologous recombination
Definition
a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence
CRISPR/Cas9
True or False:
Germline integration is not well developed in virus vector technology
True
Definition
a pathway that repairs double-strand breaks in DNA
Non-homologous end joining
Definition
any nucleobase followed by two guanine bases that are recognised as the PAM sequence by Cas9 molecules during CRISPR/Cas9
NGG
How does pronuclear injection work?
This procedure involves collecting fertilized eggs at the single cell stage. For a brief window of time the pronuclei containing the genetic material from the sperm head and the egg are visible within the protoplasm. At this stage, a linearized DNA construct is injected into one of the pronuclei. The injected eggs are then transferred into the oviducts of pseudopregnant foster mice. Generally 10 to 20% of the pups born to the foster mothers have integrated the injected DNA into their genomes, thus becoming transgenic.
Definition
proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species
Transcription activator-like effectors (TALE)
What are the advantages of gene knockouts?
- Can knockout the target gene
- Or mutate or knock in new gene
How can the transcriptome be targeted through reverse genetics strategies?
- Overexpression of a gene, e.g. overexpressing more of a gene by adding a gene under control of a promoter
- Block gene expression: iRNA/knockdown (transient and non-specific)
Define
ZNF nuclease
a site-specific endonuclease designed to bind and cleave DNA at specific positions
Define
Pronuclear injection
a very common method used to create transgenic mice. This procedure involves collecting fertilized eggs at the single cell stage. For a brief window of time the pronuclei containing the genetic material from the sperm head and the egg are visible within the protoplasm. At this stage, a linearized DNA construct is injected into one of the pronuclei. The injected eggs are then transferred into the oviducts of pseudopregnant foster mice
What sites do Cre recombinase target?
loxP sites
True or False:
Pronucear injection involves the random integration of DNA
True
How do morpholinos work?
Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA).
Define
gRNA
the RNAs that guide the insertion or deletion of uridine residues into mitochondrial mRNAs in kinetoplastid protists in a process known as RNA editing
Definition
a very common method used to create transgenic mice. This procedure involves collecting fertilized eggs at the single cell stage. For a brief window of time the pronuclei containing the genetic material from the sperm head and the egg are visible within the protoplasm. At this stage, a linearized DNA construct is injected into one of the pronuclei. The injected eggs are then transferred into the oviducts of pseudopregnant foster mice
Pronuclear injection
Define
TALENS
restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands)
Define
PAM site
a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system
Define
Non-homologous end joining
a pathway that repairs double-strand breaks in DNA
What is the main issue with pronuclear injection?
Random insertion into the genome can disrupt a gene resulting in the wrong phenotype being expressed
Define
Forward genetics
an approach used to identify genes (or set of genes) responsible for a particular phenotype of an organism
Define
Morpholino
a type of oligomer molecule used in molecular biology to modify gene expression. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA).
What are the advantages of conditional knockouts?
- Can knockout the target gene
- Can study embryonic lethal genes
- Great for studying tissue/cell type specific effects
How can you perform targeted insertion/deletion of a gene to study reverse genetics?
- Knockouts
- Knockins
- Gene editing
Define
RNA interference (RNAi)
a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules
What are the two possible mechanisms of repair when using zinc finger nucleases?
Non-homologous end joining
Homology-directed repair
What does RNA interference target?
Endogenous RNA
Definition
a small protein structural motif that is characterized by the coordination of one or morezinc ions (Zn2+) in order to stabilize the fold
Zinc finger (ZF) proteins
Definition
a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a single-stranded RNA (ssRNA) fragment, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA).
RNA-induced silencing complex (RISC)