Gene Technology Flashcards
Describe transfection and translation and highlight their pros and cons.
Transfection is the process of introducing foreign DNA into cells, while translation is the process of synthesizing proteins from mRNA. Transfection can be used to study gene function, expression, and regulation, as well as to produce recombinant proteins or gene therapy vectors. Translation can be used to produce proteins for research or therapeutic purposes, as well as to study protein structure and function. Some pros and cons of transfection are:
Pros: Allows manipulation of gene expression and function in various cell types; can be transient or stable; can introduce multiple genes or gene variants; can use different methods and reagents depending on the cell type and the goal.
Cons: Can be inefficient, toxic, or non-specific; can cause unwanted effects on cell physiology or gene regulation; can be affected by cell cycle, culture conditions, or DNA quality; can be expensive or time-consuming. Some pros and cons of translation are:
Pros: Allows production of proteins in their native or modified forms; can use different expression systems (bacterial, yeast, insect, mammalian, etc.) depending on the protein properties and the goal; can be scaled up or down; can be coupled with purification and characterization techniques.
Cons: Can be affected by codon usage, mRNA stability, or protein folding; can require optimization of expression conditions, vectors, or hosts; can produce insoluble, inactive, or unstable proteins; can be expensive or time-consuming.
Describe the main techniques used for DNA transfection, high lightening their pros and
cons.
The main techniques used for DNA transfection are:
Microinjection: Naked DNA is directly injected into the cell nucleus using a needle. It is mostly used for inserting DNA into cells with large nuclei, such as newly fertilized eggs.
Pros: High efficiency and specificity; allows direct visualization of the injection; can introduce large DNA fragments.
Cons: Requires special equipment and training; low throughput; can cause damage or death to the cells; can be affected by DNA concentration and viscosity.
Transfection: Cells are exposed to the naked DNA using physical or chemical methods that facilitate the uptake of DNA across the cell membrane. It can be used for various cell types and DNA sizes. Some common methods are:
Nucleofection: High-voltage pulses of electricity create temporary pores in the cell membrane that allow the exogenous DNA to enter the cell.
Pros: Fast, efficient, and reproducible; can transfect hard-to-transfect or non-dividing cells; can introduce multiple genes or gene variants.
Cons: Expensive; can cause cell death or stress; requires optimization of the pulse parameters and the DNA amount.
Lipofection: DNA is packaged in a positively-charged vesicle that fuses with the cell membrane and releases the DNA into the cytoplasm.
Pros: Fast, relatively cheap, and relatively efficient and reproducible; can transfect various cell types; can introduce multiple genes or gene variants.
Cons: Efficiency is cell type-dependent and usually not very high; usually transient (no permanent integration into the host genome); can cause cytotoxicity or immune response; requires optimization of the liposome-to-DNA ratio and the culture medium.
Calcium phosphate: Positively-charged calcium phosphate precipitates the DNA and brings it close to the cell membrane, where it is taken up by endocytosis.
Pros: Fast, cheap, and simple; can transfect various cell types; can introduce large DNA fragments or multiple genes.
Cons: Not very efficient or reproducible; can cause cell death or stress; sensitive to pH and temperature changes; requires optimization of the calcium phosphate-to-DNA ratio and the culture medium.
Viral transduction: Cells are exposed to DNA packed inside viral particles that infect the cells and deliver the DNA into the nucleus. It can be used for various cell types and DNA sizes, and can achieve stable and long-term expression. Some common vectors are:
Adenovirus: Single-stranded linear DNA-based, < 10 Kb long. Targets dividing and non-dividing cells. High expression but transient (no permanent integration into the host genome).
Pros: High efficiency and specificity; easy to produce and purify; can transduce various cell types and tissues; can introduce large DNA fragments or multiple genes.
Cons: Expensive; can cause immune response or inflammation; can be affected by pre-existing immunity; requires optimization of the multiplicity of infection (MOI) and the culture medium.
Lentivirus: RNA-based, HIV-derived, < 8 Kb long. Targets dividing and non-dividing cells. High expression and long-term expression (integrate permanently into the host genome).
Pros: High efficiency and specificity; stable and long-term expression; can transduce various cell types and tissues, including stem cells and neurons; can introduce multiple genes or gene variants.
Cons: Expensive; can cause insertional mutagenesis or oncogenesis; requires biosafety level 2 or 3 facilities; requires optimization of the MOI and the culture medium.
Retrovirus: RNA-based, Murine Leukemia virus-derived, < 8 Kb long. Targets only dividing cells. High expression and long-term expression (integrate permanently into the host genome).
Pros: High efficiency and specificity; stable and long-term expression; can transduce various cell types and tissues; can introduce multiple genes or gene variants.
Cons: Expensive; can cause insertional mutagenesis or oncogenesis; requires biosafety level 2 or 3 facilities; requires optimization of the MOI and the culture medium; cannot transduce non-dividing cells.
Describe the most used vectors for viral transduction, high lightening their pros and cons.
Viral transduction is a method of DNA delivery that uses DNA packed inside viral particles (very efficient)1.
The most used vectors for viral transduction are:2
Adenovirus: a single strand linear DNA-based virus that can target dividing and non-dividing cells3. It has high expression but transient (no permanent integration into the host genome). It can carry up to 10 Kb of foreign DNA.
Lentivirus: an RNA-based virus derived from HIV that can target dividing and non-dividing cells4. It has high expression and long term expression (integrate permanently). It can carry up to 8 Kb of foreign DNA.
Retrovirus: an RNA-based virus derived from Murine Leukemia virus that can target only dividing cells4. It has high expression and long term expression (integrate permanently). It can carry up to 8 Kb of foreign DNA.
The pros and cons of viral transduction are:
Pros: high efficiency, stable expression, wide range of cell types.
Cons: biohazard risk, expensive, slow, multi-steps, limited payload size, possible immune response, possible insertional mutagenesis5.
Adenovirus:
Pros: targets dividing and non-dividing cells; high expression level.
Cons: transient expression (no integration); immune response; limited payload size (< 10 Kb).
Lentivirus:
Pros: targets dividing and non-dividing cells; high expression and long-term expression (integration); low immunogenicity.
Cons: biosafety risk (HIV-derived); limited payload size (< 8 Kb).
Retrovirus:
Pros: targets only dividing cells; high expression and long-term expression (integration); low immunogenicity.
Cons: biosafety risk (MLV-derived); limited payload size (< 8 Kb); insertional mutagenesis.
Describe RNA interferences. What is the difference between siRNA and shRNA? Which
one would be better for long term knock down of a gene X?
RNA interference (RNAi) is a cellular mechanism that silences or knocks down the expression of a target gene by degrading its mRNA. RNAi can be induced by introducing double-stranded RNA (dsRNA) molecules that are complementary to the target mRNA sequence. There are two main types of dsRNA molecules used for RNAi:
Small interfering RNAs (siRNAs): 21 bp RNA duplexes with 2-nt 3’ overhangs. Introduced directly into the cells by transfection and accumulate in the cytoplasm.
Pros: Fast and cheap; can target multiple genes or gene variants; can achieve high knockdown efficiency.
Cons: Transient knockdown (unstable RNA); can cause off-target effects (due to high concentration or partial complementarity); requires optimization of the transfection conditions and the siRNA concentration and design.
Short hairpin RNAs (shRNAs): Hairpin structures with a stem region of paired antisense and sense strands (19-21 bp) connected by unpaired nucleotides (7-9 bp) forming a loop. Introduced as DNA or RNA in a vector by transfection or transduction, integrate into the genome, where they are transcribed and processed into siRNAs.
Pros: Stable knockdown (long-term effect); more specific; can target multiple genes or gene variants; can achieve high knockdown efficiency.
Cons: Time-consuming and expensive; can cause off-target effects (due to saturation of the RNAi machinery or partial complementarity); requires optimization of the vector design and delivery and the shRNA concentration and design.
For long-term knockdown of a gene X, shRNA would be better than siRNA, because shRNA can integrate into the genome and provide stable and long-term expression of the dsRNA, while siRNA is degraded quickly and needs to be replenished frequently.
Explain the key component required for a CRISPR/Cas9-mediated knock out of gene A.
What should you look for in the gene sequence?
The key component required for a CRISPR/Cas9-mediated knock out of gene A is a single guide RNA (sgRNA) that is complementary to a 20-bp sequence in the gene A, adjacent to a protospacer adjacent motif (PAM) sequence (usually NGG for Cas9). The sgRNA guides the Cas9 nuclease to the target site, where it cleaves both strands of the DNA, creating a double-strand break (DSB). The DSB can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR), depending on the presence of a donor template. NHEJ can introduce insertions or deletions (indels) that disrupt the gene function, while HDR can introduce precise modifications or replacements of the gene. To design a sgRNA for gene A, one should look for a 20-bp sequence that is unique, specific, and efficient for the gene A, and that is followed by a PAM sequence. One should also avoid sequences that have off-target effects, such as those that are similar to other genomic regions or that have low GC content.
Which technique would you use to insert a point mutation in gene A? Explain your choice.
To insert a point mutation in gene A, one could use CRISPR/Cas9 with a donor template that contains the desired mutation. The donor template should have homology arms that match the genomic sequence flanking the target site. The CRISPR/Cas9 system will create a DSB at the target site, and the donor template will be used as a template for HDR, resulting in the insertion of the point mutation. This technique allows precise and specific genome editing, but it requires the design and delivery of both the sgRNA and the donor template, and it depends on the efficiency and fidelity of the HDR mechanism.
If you want to study the function of gene C, but you are not sure of its essentiality, how
would you do it? Explain your choice.
If one wants to study the function of gene C, but is not sure of its essentiality, one could use RNAi to knock down its expression. RNAi does not modify the genome, but reduces the mRNA and protein levels of the target gene. This can reveal the phenotypic effects of the gene loss or reduction, without causing lethal effects or permanent changes. RNAi can also be used to test different levels of gene knockdown, by varying the concentration or the duration of the siRNA or shRNA treatment. If the gene C is essential, a partial knockdown might still allow the cell to survive and function, while revealing the phenotypic effects of the gene reduction. If the gene C is not essential, a complete knockdown might show a stronger phenotype or uncover a redundant function with other genes.
Why for curing a genetic disease targeting somatic cells is more accepted than targeting
germ-line tissues?
Targeting somatic cells is more accepted than targeting germ-line tissues for curing genetic diseases because:1
Ethical and social considerations: Germ-line modifications can be passed on to future generations, which raises concerns about consent, equity, and unforeseen consequences. Somatic modifications are limited to the individual and do not affect the germ line.
Technical and safety considerations: Germ-line modifications require early intervention (e.g., in vitro fertilization or embryo injection), which is more invasive and risky than somatic interventions (e.g., gene therapy or cell therapy). Somatic modifications can also be targeted to specific tissues or cells, reducing off-target effects and systemic toxicity.
What is the revolutionary discovery published in the Jinek et al., Science, 2012 paper?
The revolutionary discovery published in the Jinek et al., Science, 2012 paper is the demonstration that the CRISPR/Cas9 system from bacteria can be programmed with a single guide RNA (sgRNA) to cleave specific DNA sequences in vitro. This discovery paved the way for the development of CRISPR/Cas9 as a powerful and versatile tool for genome editing in various organisms and applications, such as research, medicine, agriculture, and biotechnology. The CRISPR/Cas9 system allows precise and specific genome editing, but it requires the design and delivery of both the sgRNA and the Cas9 nuclease, and it depends on the efficiency and fidelity of the DSB repair mechanisms. It also raises ethical and social considerations about the potential misuse or unforeseen consequences of genome editing.
