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)
What are the Pros of CRISPR/Cas9?
- Simple to design
- Very high efficiency of mutation
- Micro-injected directly into the zygote rather that into ES cells
- Multiple mutations can be introduced simultaneously
Definition
a gene or genetic material that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another
Transgene
Definition
one of a number of genetically-engineered adenoviruses designed to insert a gene of interest into a eukaryotic cell where the gene of interest is subsequently expressed. Unlike most other vectors, they have the ability to infect post-mitotic cells. Thus, these agents are especially useful for gene transfer into neuronal cells.
Adenovirus vector
Define
Transient knockdown
a temporary change in gene expression that does not modify the chromosomal DNA
How do you make a knockout mouse?
- In vitro mutation of the target gene in ES cells
- ES cells containing a mutated gene being studied are introduced into mouse blastocysts
- Chimeric mice contain tissues derived from either the transplanted ES cells (white) or the host cells (brown)
- These cells can contribute to both the germ-cell and somatic-cell populations
- Chimeric mice are mated to assess whether the mutation is incorporated into the germline
- Heterozygous mice for the gene mutation are mated to produce mice that are homozygous for the mutated gene
Definition
a site-specific endonuclease designed to bind and cleave DNA at specific positions
ZNF nuclease
Define
Adenovirus vector
one of a number of genetically-engineered adenoviruses designed to insert a gene of interest into a eukaryotic cell where the gene of interest is subsequently expressed. Unlike most other vectors, they have the ability to infect post-mitotic cells. Thus, these agents are especially useful for gene transfer into neuronal cells.
Definition
describes the physical co-localization of genetic loci on the same chromosome within an individual or species
Synteny
Define
NGG
any nucleobase followed by two guanine bases that are recognised as the PAM sequence by Cas9 molecules during CRISPR/Cas9
Definition
an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria
Cas9
Definition
a mouse whose DNA has been genetically engineered so that it does not express particular proteins
Knockout mice
Definition
the RNAs that guide the insertion or deletion of uridine residues into mitochondrial mRNAs in kinetoplastid protists in a process known as RNA editing
gRNA
How do you make a knockin mouse?
Step 1: A DNA construct containing a specific gene such as mutated or reporter oncogene is taken.
Step 2: These genes with flanking sequence (also called loxP) undergo inverse recombination when they are exposed to the sites carrying Cre-recombinase enzyme. This results in the omission of intervening DNA.
Step 3: Then the modified gene is introduced to the endogenous locus.
Step 4: Embryonic stem cells carrying the modified gene is inoculated into a cavity of an early mouse embryo. This is then implanted into a surrogate female mouse, where the mouse embryo matures into a chimeric mouse with germ line carrying the transgenic gene.
Step 5: The offspring of this chimeric mouse carry the gene knockin.
What viral vector is HIV?
Lentivirus
Definition
the sum total of all the messenger RNA molecules expressed from the genes of an organism
Transcriptome
True or False:
Cre recombinase enzyme can target specific tissues by controlling where it is expressed
True
Define
Gene editing
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
Definition
a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules
RNA interference (RNAi)
Definition
a temporary change in gene expression that does not modify the chromosomal DNA
Transient knockdown
What are the disadvantages of gene knockouts?
- Occurrence of random insertion still remains
- This can have a major effect on the phenotype
- Predicted that 15-40% of genes knocked out will lead to a lethal at the embryo stage
- Difficult to study the protein at different stages of development
- Neomycin resistance genes can influence the phenotype
What are the disadvantages of morpholinos?
- Limited to mostly frog and fish embryo work
- Limited lifespan (days)
- Need to be able to monitor blockage of translation using specific antibody
- Care needs to be taken to check for non-specific interaction with other mRNA “off-target effects”
- Amount of MO delivered into the cell can vary
What are alternatives to pronuclear injection?
Adenovirus vectors
Lentivirus vector
How do you make a conditional knockout?
The most commonly used technique is the Cre-lox recombination system. The Cre recombinase enzyme specifically recognizes two lox (loci of recombination) sites within DNA and causes recombination between them. During recombination two strands of DNA exchange information. This recombination will cause a deletion or inversion of the genes between the two lox sites, depending on their orientation. An entire gene can be removed to inactivate it.
How can you add a gene to the germline to study reverse genetics?
- Pronuclear injection
- Virus vectors
Define
RNA-induced silencing complex (RISC)
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).
Define
Conditional knockout
a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life
How does RNAi work?
During RNAi, long dsRNA is cut or “diced” into small fragments ~21 nucleotides long by an enzyme called “Dicer”. These small fragments, referred to as small interfering RNAs (siRNA), bind to proteins from a special family: the Argonaute proteins. After binding to an Argonaute protein, one strand of the dsRNA is removed, leaving the remaining strand available to bind to messenger RNA target sequences according to the rules of base pairing. Once bound, the Argonaute protein can either cleave the messenger RNA, destroying it, or recruit accessory factors to regulate the target sequence in other ways
How does CRISPR/Cas9 allow genome editing?
Cas9 is a nuclease that recognises the PAM site. Specific gRNA is used to target specific sequences and combine with the Cas9. The Cas9 creaes a targeted double-stranded break allowing for non-homologous end joining or homolog-directed repair
Definition
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).
Morpholino
Definition
a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life
Conditional knockout
Define
Reverse genetics
a method that is used to help understand the function of a gene by analyzing the phenotypic effects of specific engineered gene sequences
Definition
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)
TALENS
Define
Lentivirus vector
a method by which genes can be inserted, modified, or deleted in organisms using lentivirus
Define
Zinc finger (ZF) proteins
a small protein structural motif that is characterized by the coordination of one or morezinc ions (Zn2+) in order to stabilize the fold
How do TALENs allow genome editing?
The TALE region is fused to a non-specific endonuclease which creates a double-strand break. Can results in non-homologous end joining (gene disruption) or homology-directed repair (gene correction/addition)
Definition
a mechanism in cells to repair double strand DNA lesions. The most common form of HDR is homologous recombination
Homology-directed repair
How do you produce ES cells with a gene knockout?
Define
CRISPR/Cas9
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
Define
Knockout mice
a mouse whose DNA has been genetically engineered so that it does not express particular proteins
Definition
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
Knockins
Define
Synteny
describes the physical co-localization of genetic loci on the same chromosome within an individual or species
Define
Gene targeting
a genetic technique that uses homologous recombination to modify an endogenous gene
Definition
a genetic technique that uses homologous recombination to modify an endogenous gene
Gene targeting
Definition
an approach used to identify genes (or set of genes) responsible for a particular phenotype of an organism
Forward genetics
Define
Transgenesis
the introduction of a transgenes that has the potential to change the phenotype of an organism
A system that can conditionally express the knockout phenotype at different stages of development and/or in tissues is called a ___________ allele
Conditional
True or False:
Cre recombinase can allow mice to pass through the development stage without effects
True
Definition
a method by which genes can be inserted, modified, or deleted in organisms using lentivirus
Lentivirus vector
What makes morpholinos more stable than RNAi?
They are resistent to nucleases
How do you check that a knockout has worked properly?
Sequencing site on genome
Southern blot
Loss of protein expression
Define
Transgene
a gene or genetic material that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another
What are the Cons of CRISPR/Cas9?
- Off-site effects in the genome
- Can result in mosaic insertion if zygite injection method used
- Variation in the actual site and sequence of mutation
- Need to genome sequence
- Use multiple genome edited organisms
- Requires outbreeding to achieve single mutation
Define
Cas9
an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria
How do zinc finger nucleases allow genome editing?
Zinc finger proteins specifically recognize the DNA sequence. The ZF region is fused to a non-specific endonuclease which creates a double strand break. The donor DNA is added and incorporates via homology-directed repair
Definition
a method that is used to help understand the function of a gene by analyzing the phenotypic effects of specific engineered gene sequences
Reverse genetics
What are the disadvantages of conditional knockouts?
- Can take longer to get a result
- Requires a lot of specific crossing with different mice
- $$$
Define
Transcriptome
the sum total of all the messenger RNA molecules expressed from the genes of an organism
