Plasmids

what are vectors?

DNA molecules used to carry and deliver foreign DNA sequences into host cells.
Crucial in genetic engineering and gene expression studies

what are the functions of vectors?

Serve as vehicles for transporting foreign DNA into host cells.
Act as carriers for efficient delivery of desired DNA sequences.
Contain specific genetic elements for replication, propagation, and expression in host cells.

what is the importance of vectors for DNA delivery?

Vectors efficiently deliver foreign DNA into mammalian cells.
Overcome the challenge of introducing DNA directly into cells due to complex cellular membranes.
Utilize specific mechanisms like viral-based vectors or non-viral delivery systems

what is the importance of vectors for regulatory elements?

Vectors contain promoters, enhancers, and transcription termination signals to
ensure proper gene expression.
Promoters initiate gene transcription, while enhancers modulate promoter activity
Inclusion of appropriate regulatory elements allows for precise control of gene expression levels and cell-specific expression

what is the importance of vectors as selection markers?

vectors incorporate selectable markers like antibiotic resistance genes or fluorescent proteins.
Enable identification and selection of cells that have taken up the vector and expressed the desired gene.
Provide a way to distinguish between integrated and non-integrated cells, allowing for isolation and enrichment of cells with desired gene expression

what is the importance of vectors in expression systems?

Vectors can be designed to work with specific expression systems in mammalian cells.
Engineered for inducible expression systems, allowing precise temporal and spatial control of gene expression through the addition or removal of specific inducers

what are the roles of promoters?

Determine when and where a gene is expressed
Located upstream of the coding sequence.
Serve as binding sites for RNA polymerase, initiating transcription

what is the importance of promoters in vectors?

Regulate gene expression levels and direct expression in specific cell types or conditions.
Varying strengths influence mRNA and protein expression levels.
Strong promoters generate high mRNA and protein levels, while weak promoters result in lower expression.

what are constitutive promoters?

Drive gene expression consistently and at relatively high levels in most cell types.
Examples: CMV (cytomegalovirus) promoter, SV40 (simian virus 40) promoter.
Commonly used for constant expression in basic research or when generating stable cell lines

what are tissue-specific promoters?

Drive gene expression in specific cell types or tissues.
Derived from genes normally expressed in those specific cells.
Allow targeted expression, useful in gene therapy or developmental biology studies

what are inducible promoters?

Allow control over gene expression by responding to specific inducers or environmental stimuli.
Regulate gene expression based on the presence or absence of the inducer.
Examples: tetracycline-inducible promoter (Tet-On/Tet-Off system), ecdysone inducible promoter.
Offer temporal and spatial control over gene expression, facilitating studies requiring precise regulation or dynamic expression patterns.

what is codon usage in codding sequences?

Refers to the frequency of specific codons in the DNA sequence.
Using host organism-preferred codons enhances translation efficiency.
Optimizing codon usage can improve protein expression levels

what is mRNA stability in coding sequences?

mRNA stability impacts protein expression levels.
Designing stable secondary structures or avoiding degradation-prone sequences increases mRNA stability and enhances protein expression

what is the Kozak sequence?

Specific sequence surrounding the start codon (usually AUG) that enhances translation initiation.
Including an optimized Kozak sequence improves translation efficiency and protein expression levels

what are fusion tags?

Added to aid in protein purification, detection, or localization.
Common fusion tags: polyhistidine (His-tag), glutathione S-transferase (GST), green fluorescent protein (GFP).

what is the purpose of selection markers?

Enable identification and selection of cells that have taken up the vector and expressed the gene of interest.
Distinguish transformed cells from non-transformed cells or those that have not integrated the vector

what are antibiotic resistance genes?

Encode proteins providing resistance to specific antibiotics
Transformed cells expressing these genes survive in the presence of corresponding antibiotics, while non-transformed cells are eliminated

what are fluorescent proteins?

Genes encoding fluorescent proteins like green fluorescent protein (GFP) serve as
selection markers.
Cells expressing fluorescent proteins can be easily identified and sorted using fluorescence-activated cell sorting (FACS) or fluorescence microscopy

what are reporter genes?

Encode enzymes producing detectable signals like colour change or luminescence when expressed.
Enable visualization or quantification of gene expression in transformed cells.

what are viral vectors for gene expression?

Derived from viruses and extensively used in gene expression studies and gene
therapy applications.
Efficient delivery of genetic material into host cells due to their natural cell- infecting ability

what are lentiviral vectors?

Derived from lentiviruses, capable of infecting both dividing and non- dividing cells.
Integration into host genome - results in stable, long-term gene expression.
Large cargo capacity
Can accommodate large DNA sequences, including full genes or gene clusters.

what are the applications of lentiviral vectors?

Gene therapy for inherited disorders, cancer therapies, and genetic diseases.
Transgenic animal production.
Basic research for studying gene function, regulation, and protein expression in various cell types

what are the applications of plasmid vectors?

Easy manipulation and production: Easily manipulated in the laboratory and produced in large quantities using bacterial culture systems.
Transient gene expression: Typically do not integrate into the host genome, resulting in transient gene expression.
Gene therapy applications: Explored in gene therapy approaches, particularly for ex vivo gene therapy.
DNA vaccines: Used to deliver specific genes encoding antigens for stimulating an immune response against pathogens.
Gene knockout studies: Used for gene knockout studies by delivering CRISPR/Cas9 components or other gene-editing tools to target specific genes.

what is electroporation?

Involves the application of electric pulses to cells, creating temporary pores in the cell
membrane, allowing DNA to enter the cells.
Widely used non-viral method for gene delivery, particularly in research laboratories.

what are nanoparticle-based vectors?

Nanoparticles functionalized with DNA used for gene delivery, imaging, or theranostic
applications
Examples include gold nanoparticles or quantum dots

what are polymer-based vectors?

Offer efficient DNA delivery and can be modified for improved targeting or controlled
release of genes.
Examples include polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA)

what are the steps in transduction mechanisms?

Cell attachment: Viral vector binds to specific receptors on the cell surface, facilitating
its entry into the target cell.
Internalization: Taken up by the cell through mechanisms like receptor-mediated
endocytosis or direct fusion with the cell membrane.
Escape from endosomes: If internalized via endocytosis, some viral vectors need to escape from endosomes to avoid degradation in lysosomes, facilitated by pH sensitive viral proteins or viral proteins that disrupt endosomal membranes.
Nuclear localization: Viral vector or its genetic material must reach the cell nucleus for
gene expression, facilitated by nuclear localization signals.
Gene expression: Genetic material is released and can integrate into the host genome or undergo transcription and translation to produce the desired protein.

what are the advantages of viral-mediated gene delivery?

High transduction efficiency: Viral vectors have evolved to efficiently infect and
deliver genes into host cells, resulting in high transduction efficiency.
Stable and long-term gene expression: Integrating viral vectors, such as lentiviral vectors, can integrate their genetic material into the host genome, leading to stable and long-term gene expression.
Cell type specificity: Some viral vectors can be engineered to target specific cell types by modifying their surface proteins or using cell-specific promoters.
Large cargo capacity: Viral vectors can accommodate relatively large DNA sequences, allowing the delivery of full-length genes or gene clusters.

what are the limitations of viral-mediated gene delivery?

Immunogenicity - Viral vectors can trigger immune responses in the host, potentially leading to inflammation or immune rejection.
Safety Concerns - Risk of insertional mutagenesis, where the integration of viral DNA into the host genome can disrupt normal gene function, potentially leading to adverse effects.
Limited Cargo Capacity - Although relatively large compared to non-viral vectors, viral vectors are still limited in the size of DNA they can carry.
Manufacturing Challenges - More challenging and costly to produce at large scales compared to non-viral vectors.

what are the principles of lipofection?

Formation of lipid-DNA complexes: Cationic lipids interact with DNA, forming lipid-
DNA complexes or lipoplexes.
Cellular uptake: Lipoplexes are taken up by cells through endocytosis or direct fusion with the cell membrane.
Endosomal escape: Lipoplexes may need to escape from endosomes to avoid degradation in lysosomes, facilitated by pH-sensitive lipids or endosome-disrupting peptides.
Nuclear localization: Lipoplexes must reach the nucleus for gene expression, aided by nuclear localization signals.
Gene expression: Once inside the nucleus, the DNA undergoes transcription and translation to produce the desired protein.

what are the applications of lipofection?

Transient gene expression: Commonly used for transient gene expression experiments.
Gene silencing: Delivery of siRNA or shRNA for gene knockdown studies or therapeutic applications.
Gene editing: Delivery of gene-editing tools, such as CRISPR/Cas9 components, for targeted genome modifications

what are the principles of electroporation?

Preparation of cells: Cells are suspended in a conductive buffer and mixed with DNA.
Electroporation pulse application: Cells are subjected to a brief, high-voltage electric pulse, creating temporary pores in the cell membrane.
DNA uptake: The temporary pores allow DNA to enter the cells.
Cell recovery: After electroporation, cells recover and express the introduced DNA

what are the applications of electroporation?

Transient gene expression - Often used for transient gene expression studies, allowing introduced genes to be expressed for a limited period.
Gene editing - Delivery of gene-editing tools, such as CRISPR/Cas9 components, for efficient genome modifications.
Cell transformation - Commonly employed in bacterial and yeast transformation to introduce foreign DNA into these cells.
Cell-based therapies - Used in ex vivo gene therapy approaches, where cells are removed from the body, transfected with the gene of interest, and then re-introduced into the patient.

how does viral and non-viral vectors differ?

Advantages of Viral Vectors
Efficient gene delivery into host cells.
Long-term gene expression through integration into the host genome.
Suitable for in vivo gene therapy applications.
Disadvantages of Viral Vectors
Immunogenicity and potential safety concerns.
Limited cargo capacity and manufacturing challenges.
Advantages of Non-Viral Vectors
Generally safer with lower immunogenicity.
Flexible in cargo capacity and relatively easier to manufacture at large scales.
Disadvantages of Non-Viral Vectors
Less efficient gene delivery compared to viral vectors.
Typically limited to transient gene expression. Viral vectors are efficient for long-term gene delivery, while non-viral vectors are safer and easier to produce, but less effective

what is the role of enhancers in vectors?

Enhancers are regulatory elements found in DNA sequences that play a crucial
role in enhancing gene expression

what are the main functions of enhancers in vectors?

Increasing Transcriptional Activity
Facilitate the recruitment of transcription factors and RNA polymerase II to the promoter region, leading to increased transcriptional activity of the gene.
Cell-Specific Gene Expression
Confer cell-type specificity to gene expression, allowing the targeting of gene expression to desired cell populations.
Enhancing Expression Levels
Increase the transcriptional activity of the gene, leading to higher levels of gene expression, which may be important for achieving therapeutic effects in gene therapy applications.
Enhancing Temporal Expression
Some enhancers are regulated by specific signals or conditions, allowing for temporal control of gene expression, which can be useful in applications requiring precise timing of gene expression.

what are the main functions of terminators?

mRNA Processing
Contain specific sequences that signal the end of the gene's coding region, recognized by molecular machinery involved in mRNA processing, including cleavage and polyadenylation factors. Proper mRNA processing is essential for the stability and functionality of the mRNA molecule.
Transcriptional Termination
Facilitate the termination of transcription by helping to release the RNA polymerase complex from the DNA template, ensuring that transcription stops at the appropriate site and prevents read-through transcription into neighbouring genes.
mRNA Stability
Contribute to mRNA stability by proper termination and polyadenylation, protecting it from degradation. The addition of a poly(A) tail at the 3' end of mRNA provides stability and aids in efficient translation of the mRNA into protein.

what are the importance of selection markers in identifying transformed cells?

Selection markers play a crucial role in identifying and isolating cells that have successfully taken up and expressed the gene of interest delivered by a vector.
These markers provide a selectable phenotype to transformed cells, allowing for their isolation and enrichment.
The use of selection markers is particularly important in cases where the efficiency of gene delivery is low, and a small fraction of cells acquire and express the gene

what are the reasons for incorporating selection markers in vectors?

Enrichment of Transformed Cells
Enables the identification and enrichment of cells that have taken up and expressed the vector, crucial when the efficiency of gene delivery is low.
Discrimination from Untransformed Cells
Provides a clear distinction between transformed cells and untransformed cells in a mixed population, especially when the gene of interest does not confer a readily distinguishable phenotype.
Confirmation of Successful Gene Transfer
Serves as an indicator of successful gene transfer and expression, confirming that the gene of interest has been delivered and is functional.

what are some key considerations for designing and constructing vectors for gene expression?

Vector Backbone
Serves as the structural framework for the gene of interest and other components, including origins of replication, selectable markers, and cloning sites.
Promoters
DNA sequences that drive gene expression, determining timing, strength, and cell type specificity.

Enhancers and Regulatory Elements
Enhancers can increase promoter activity, while regulatory elements like
insulators can modulate or suppress gene expression, allowing precise control.
Polyadenylation Signal
Crucial for mRNA stability and proper termination of transcription, ensuring the
production of stable and functional mRNA transcripts.
Selection Markers
Important for identifying and enriching transformed cells based on the
mechanism of action and target cell type.
Sequencing and Cloning Sites
Essential for vector construction and manipulation, allowing for insertion,
verification of the gene of interest, and other modifications.

what are the considerations for vector design in specific applications?

Gene Therapy
Protein Production
Functional Genomics

Cell Lineage Tracing
Inducible Gene Expression
Tissue-Specific Expression