DEEL 7 Genanalyse in eukaryoten en RNAi.docx

# Gene transfer methods in eukaryotic cells Gene transfer methods in eukaryotic cells provide crucial tools for molecular biology research and biotechnology, enabling the introduction of foreign genetic material into these complex cellular systems for various applications. ## 1. Gene transfer methods in eukaryotic cells ### 1.1 Introduction to gene transfer Gene transfer encompasses techniques used to introduce foreign DNA or RNA into eukaryotic cells. This process is fundamental for studying gene function, developing therapeutic strategies, and engineering organisms. The primary methods include transfection, transduction, and transformation, each with distinct mechanisms, applications, and outcomes. ### 1.2 Transfection Transfection is the process of introducing nucleic acids (DNA or RNA) into eukaryotic cells by non-viral methods. It can result in either transient or stable expression of the introduced genetic material. #### 1.2.1 Transient transfection Transient transfection leads to the temporary expression of the foreign genetic material, typically lasting for 24 to 72 hours. The introduced DNA does not integrate into the host cell's genome and is eventually lost during cell division. This method is useful for short-term gene expression studies. * **Process:** A vector containing the gene of interest is introduced into actively dividing cells using methods like electroporation or lipofection. * **Outcome:** The foreign DNA exists episomally (extrachromosomally) within the cell, leading to temporary protein production. * **Limitations:** Approximately 50% of cells may not be successfully transfected, and there is no permanent genetic modification or selection for transfected cells. #### 1.2.2 Stable transfection Stable transfection results in the permanent integration of the foreign genetic material into the host cell's genome. This allows for long-term, heritable expression of the introduced genes, which is essential for creating stable cell lines. * **Process:** Similar to transient transfection, a vector is introduced into cells. However, this is followed by a selection step to isolate cells that have successfully integrated the foreign DNA. * **Outcome:** The foreign DNA integrates randomly into the host genome, potentially leading to overexpression if multiple copies concatenate. This stable integration results in permanent expression of the introduced gene. * **Selection:** Selection systems are crucial for identifying and isolating stably transfected cells. #### 1.2.3 Transfection techniques A variety of methods are employed for transfection, leveraging physical or chemical means to facilitate nucleic acid entry into cells. * **Calcium-phosphate/CaCl$_2$ precipitation:** * This method involves precipitating DNA with calcium chloride in a phosphate buffer. The precipitated DNA is then added to actively dividing cells and incubated. * It can be used for both transient and stable expression. * The process involves incubating DNA with calcium phosphate to form precipitates that cells can take up. * **Lipofection:** * This technique uses cationic lipids to form liposomes that encapsulate the negatively charged DNA. These liposomes interact with the negatively charged cell membrane, facilitating DNA entry into the cell. * It is a widely used method for both transient and stable transfections. * **Electroporation:** * Cells and DNA are mixed in a special cuvette and subjected to a brief electrical pulse. This pulse creates temporary pores in the cell membrane, allowing DNA to enter. * Can be used for transient or stable expression. * It is a physical method that does not modify the incorporated genome. * **Receptor-mediated endocytosis:** * DNA is complexed with cationic molecules, such as polylysine and transferrin. These complexes bind to specific receptors (e.g., transferrin receptor) on the cell surface and are internalized via endocytosis. * The complexes are then either delivered to lysosomes or the nucleus. * Polyethylenimine (PEI) can also be used as a cationic polymer in this approach. * **Magnetofection:** * DNA is complexed with magnetic nanoparticles. An external magnetic field is used to attract these nanoparticles to the cell surface, accelerating uptake via endocytosis. * A disadvantage is that it can sometimes damage cells. * **Nucleofection:** * A variant of electroporation where an electrical pulse opens temporary pores in the cell membrane, directly transporting DNA or RNA into the nucleus. * **Nanoblades:** * These are microscopic structures used to deliver large genetic constructs into cells. They function by physically piercing the cell membrane. ### 1.3 Transduction Transduction is a method of gene transfer mediated by viruses. Viruses act as vectors, delivering genetic material into host cells. This approach often leads to stable integration of the foreign DNA into the host genome. #### 1.3.1 Viral vectors Viral vectors are engineered viruses that have been modified to carry therapeutic genes or genes for research purposes. * **Adenovirus vectors:** * These are DNA viruses that can infect both dividing and non-dividing cells. * **Constructs:** Viral DNA is modified by excising non-essential regions ("stuffer regions") to create space for the gene of interest. Co-transfection of modified viral DNA and helper DNA can produce infectious virus particles. * **Application:** Used for transient expression and can be engineered to produce virus particles. * **SV40 (Simian virus 40) vectors:** * This DNA virus can lead to permanent constructs and transient expression. * **Application:** COS and HEK cells are used, which express the SV40 T antigen, facilitating high-copy replication of SV40-based plasmids (cos-vectors) containing the gene of interest. High copy numbers can lead to cell death due to aggregation. * **Retroviral vectors:** * These are RNA viruses that integrate their genetic material into the host cell's genome. They are effective for gene transfer into dividing cells. * **Structure:** The viral genome contains Long Terminal Repeats (LTRs), gag, pol, and env genes. For vector construction, these essential genes are removed and placed on separate packaging vectors to ensure safety (the produced virus particles cannot replicate independently). * **Process:** A transfer vector containing the gene of interest, a packaging signal ($\psi$), and LTRs is co-transfected with packaging vectors into a cell line. This produces replication-incompetent viral particles that can infect target cells. * **Integration:** Upon infection, the RNA genome is reverse transcribed into DNA, circularized, and integrated randomly into the host genome. * **Limitations:** Do not efficiently infect non-dividing cells. * **Lentiviral vectors:** * These are a subclass of retroviral vectors, based on Human Immunodeficiency Virus (HIV-1). They are highly efficient at transducing both dividing and non-dividing cells, leading to stable integration. * **Mechanism:** Similar to retroviral vectors, lentiviral vectors are produced by co-transfecting multiple plasmids encoding viral proteins and the transfer vector containing the gene of interest. The resulting viral particles infect target cells, where the RNA genome is reverse transcribed and integrated into the host genome. * **Features:** Lentiviral vectors often include elements like CMV or SV40 promoters, SV40 origin of replication (ORI), splice sites, and various tags for detection. * **SEND (Selective endogenous encapsulation for cellular delivery):** * This RNA-based system uses three plasmids to produce virus-like particles (VLPs). * **Components:** A PEG10-expressing plasmid (encodes a retrovirus-like protein for capsid formation), a cargo RNA plasmid (containing the gene of interest and PEG10 UTRs), and a fusogenic plasmid (encodes a fusion protein for cellular entry). * **Process:** Transfection into a suitable cell leads to VLP assembly and release. These VLPs then transduce target cells. * **Advantage:** RNA-based, meaning no integration into the host genome. #### 1.3.2 Determining viral titer: Multiplicity of Infection (MOI) The Multiplicity of Infection (MOI) is a critical parameter in viral transduction experiments. It is defined as the ratio of the number of infectious virus particles to the number of target cells. * **Formula:** $$ \text{MOI} = \frac{\text{# virus particles}}{\text{# cells}} $$ * **Significance:** MOI influences the efficiency of transduction and the number of integrated copies of the transgene per cell. ### 1.4 Transformation Transformation primarily refers to the process of introducing foreign DNA into bacterial cells. While the term is also sometimes used in a broader context for other organisms, its classical definition is bacterial. ### 1.5 Plant gene transfer Introducing foreign genes into plant cells requires specialized methods due to the presence of a rigid cell wall. #### 1.5.1 Plant vectors * **Ti-plasmid (Tumor-inducing plasmid):** * Used by *Agrobacterium tumefaciens* to transfer a segment of its DNA (T-DNA) into the plant genome, causing tumor formation. * **Mechanism:** *Agrobacterium* detects plant wound signals (phenols), which activate virulence (vir) genes. These genes enable the bacterium to excise a single strand of T-DNA, which then migrates into the plant cell and integrates into its genome. * **Components:** The T-DNA is flanked by Left Border (LB) and Right Border (RB) sequences. Vir genes are also essential for this process. * **Application:** Modified Ti-plasmids are used as vectors to deliver desired genes into plants. For example, genes to reduce polygalacturonase expression can lead to firmer fruit. * **Binary vector system:** * This system utilizes two plasmids: one containing the gene of interest and another that is a helper Ti-plasmid providing the necessary vir genes. This offers more flexibility and ease of manipulation. #### 1.5.2 Plant transformation techniques * **Protoplast regeneration:** * Plant cell walls are enzymatically removed to create protoplasts. * DNA can then be introduced into protoplasts via methods like polyethylene glycol (PEG) treatment or electroporation. * The modified protoplasts are then cultured to regenerate whole plants. * **Biolistics (Gene gun):** * This method uses physical force to deliver DNA-coated particles (e.g., gold or tungsten) into plant cells or tissues. ### 1.6 Selection systems for gene transfer Selection systems are essential for identifying and isolating cells that have successfully incorporated the foreign genetic material, especially in stable transfection and transduction. #### 1.6.1 Complementary selection * **Mechanism:** This method relies on restoring a lost genetic function in the recipient cell by introducing a functional copy of the gene. * **Example: Thymidine kinase (TK) deficiency:** * TK-deficient cells are used. A vector carrying a functional TK gene and another gene of interest is co-transfected. * Cells are then cultured in HAT medium (hypoxanthine, aminopterin, thymidine). Only cells that have taken up and express the functional TK gene can survive and proliferate in this medium. #### 1.6.2 Dominant selectable markers * **Mechanism:** These are genes that confer resistance to an antibiotic or toxic substance. They work independently of the recipient cell's genotype. * **Example: Neomycin resistance (NeoR):** * The neomycin resistance gene encodes an enzyme (aminoglycoside phosphotransferase, APH) that inactivates the antibiotic G418 (a neomycin analog). * Cells successfully transfected with a vector containing the NeoR gene will be resistant to G418 and can be selected for in culture medium containing G418. #### 1.6.3 Selection pressure * **Concept:** Applying a selective agent (e.g., an inhibitor like methotrexate, MTX) that targets a specific cellular pathway. If the introduced gene confers resistance or overcomes the inhibition (e.g., by overexpressing dihydrofolate reductase, DHFR), cells expressing the transgene will survive. ### 1.7 Media for cell cultures Various specialized cell culture media are used to support the growth and maintenance of eukaryotic cells during gene transfer experiments. Common examples include RPMI, Opti-MEM, EMEM (Eagle's Minimum Essential Medium), and DMEM (Dulbecco's Modified Eagle Medium). ### 1.8 RNA interference (RNAi) and post-transcriptional gene silencing RNA interference (RNAi) is a natural biological process that regulates gene expression at the post-transcriptional level, primarily by degrading specific messenger RNA (mRNA) molecules. Exogenous application of RNAi components can be used to "silence" target genes. * **Interfering vs. Degrading:** RNAi interferes with gene expression, typically by leading to mRNA degradation or translational repression, rather than directly altering the DNA sequence like CRISPR-Cas systems. #### 1.8.1 Mechanisms of post-transcriptional gene silencing * **MicroRNAs (miRNAs):** * Endogenous small RNAs that typically bind with partial complementarity to target mRNAs, leading to translational repression. * **Biogenesis:** Processed from pri-miRNA precursors in the nucleus by Drosha, then exported to the cytoplasm where Dicer cleaves pre-miRNA into a ~21-22 nucleotide duplex. This duplex is incorporated into the RNA-induced silencing complex (RISC). * **Function:** RISC uses the miRNA as a guide to find complementary sequences on target mRNAs. Partial matches generally inhibit translation. * **Small interfering RNAs (siRNAs):** * Small RNAs, typically ~21-22 nucleotides long, derived from longer double-stranded RNA (dsRNA) molecules. They are involved in gene silencing through mRNA cleavage. * **Biogenesis:** Introduced dsRNA is processed by Dicer into siRNA duplexes. These are incorporated into RISC, which uses one strand (the guide strand) to find a target mRNA. * **Function:** If there is a perfect or near-perfect complementarity between the siRNA guide strand and the target mRNA, the RISC complex (specifically, Argonaute protein within RISC) will cleave the mRNA, leading to its degradation. * **Short hairpin RNAs (shRNAs):** * These are synthetic RNA molecules designed to fold into a hairpin structure. When expressed within a cell, they are processed by the cell's own machinery (Dicer) into siRNAs, which then engage the RNAi pathway. * shRNAs are commonly used in research to achieve gene knockdown. * **Dharmancon's ON-TARGET siRNAs:** * These are modified siRNAs designed for improved target recognition and cleavage. Modifications, particularly to the sense strand, can enhance the specificity and efficacy of gene silencing. #### 1.8.2 RNAi vectors RNAi can be stably expressed within cells using specialized vectors. * **Vector-based expression of shRNAs:** * shRNA sequences are cloned into RNA expression vectors under the control of appropriate promoters (e.g., U6 promoter, which is active in vivo for RNA transcription). * These vectors can be designed for transient transfection or stable integration into the genome using retroviral elements. * **pSIREN-shuttle:** * Designed for transient transfection. Features a U6 promoter for in vivo transcription of shRNA, a multiple cloning site (MCS) for cloning the shRNA sequence, a selection marker, and a SV40 origin of replication (ORI). It lacks retroviral elements, thus no genomic integration. * **pSIREN-RetroQ:** * Designed for stable transfection. It also has a U6 promoter and MCS but includes retroviral LTRs, enabling integration into the host genome. It also contains a packaging signal for viral production. #### 1.8.3 Therapeutic applications of RNAi RNAi has significant therapeutic potential for treating various diseases by silencing disease-causing genes. * **Antisense Oligonucleotides (ASOs):** * Short, single-stranded DNA or RNA molecules that can bind to target mRNA. * **Mechanism:** ASOs can block translation, modulate splicing, or induce mRNA degradation. * **Applications:** Used for protein downregulation or alternative splicing modification. * **AntagoNats:** * These are molecules designed to block antisense transcripts, potentially leading to gene upregulation. * **Ribozymes:** * Catalytically active oligonucleotides that can promote mRNA degradation. * **Clinical examples:** * **Duchenne Muscular Dystrophy:** Antisense-mediated exon skipping is being explored to correct the genetic defect. #### 1.8.4 siRNA vs. miRNA and outcomes * **siRNA:** Typically induces cleavage and degradation of the target mRNA due to high complementarity. This leads to a strong "gene knockdown." * **miRNA:** Usually leads to translational repression due to partial complementarity. The mRNA is not necessarily degraded immediately but its translation is inhibited. #### 1.8.5 Chemical synthesis of RNAi components * **Synthetic siRNAs and ASOs:** Can be chemically synthesized and delivered directly to cells or tissues. * **In vitro transcription:** RNA molecules can also be transcribed from DNA templates in vitro. #### 1.8.6 Considerations for RNAi * **Off-target effects:** The guide strand of siRNA or miRNA can sometimes bind to unintended mRNA targets, leading to silencing of genes other than the intended one. * **On-target recognition and cleavage:** Achieving efficient and specific silencing of the intended target is crucial. * **Delivery:** Effective delivery of RNAi molecules into target cells or tissues remains a significant challenge for in vivo applications. **Key definitions:** * **miRNA:** Micro RNA, ~21 nucleotides. * **pri-miRNA:** Primary miRNA precursor. * **siRNA:** Small interfering RNA, ~21 nucleotides, derived from dsRNA. * **shRNA:** Small hairpin RNA, folded dsRNA precursor. * **dsRNA:** Double-stranded RNA. * **DICER:** Enzyme that processes dsRNA into siRNAs/miRNAs. * **RISC:** RNA-induced silencing complex, involved in gene silencing. * **ASO:** Antisense Oligonucleotide, blocks mRNA or modifies splicing. * **AntagoNAT:** Antisense-based molecule that blocks antisense transcripts. --- # Post-transcriptional control and RNA interference Post-transcriptional control mechanisms regulate gene expression after the initial transcription of DNA into RNA, with RNA interference (RNAi) being a prominent pathway that silences gene expression by targeting specific RNA molecules. ### 2.1 Principles of post-transcriptional gene silencing Post-transcriptional gene silencing involves regulating gene expression at the RNA level, primarily by degrading target messenger RNA (mRNA) or inhibiting its translation. RNA interference (RNAi) is a key mechanism within this category. ### 2.2 RNA interference (RNAi) RNAi is a conserved biological process in which RNA molecules inhibit gene expression or translation, typically by neutralizing targeted mRNA molecules. It achieves this through sequence-specific recognition and degradation or translational repression of complementary RNA. #### 2.2.1 Mechanisms of RNAi The RNAi pathway involves the generation of small RNA molecules that guide effector complexes to target RNAs. * **siRNA generation and action:** * Small interfering RNAs (siRNAs) are typically derived from longer double-stranded RNA (dsRNA) precursors. * These dsRNAs are processed by an enzyme called Dicer, which cleaves them into short fragments of 21-22 nucleotides with a 3' hydroxyl (OH) group and a 2-base overhang. * The processed siRNA is then loaded into the RNA-induced silencing complex (RISC). * Within RISC, one strand (the passenger strand) is removed, leaving the guide strand to direct RISC to complementary target mRNA sequences. * A perfect complementarity between the siRNA guide strand and the target mRNA leads to the cleavage and degradation of the mRNA. * **miRNA generation and action:** * MicroRNAs (miRNAs) are endogenously produced small regulatory RNAs that are transcribed from distinct genes. * They are initially processed in the nucleus into a precursor called pri-miRNA, which is then processed into pre-miRNA by the enzyme Drosha. * Pre-miRNA is exported to the cytoplasm and further processed by Dicer into a ~21-22 nucleotide dsRNA duplex. * Similar to siRNAs, one strand of the miRNA duplex is incorporated into RISC. * However, miRNAs typically exhibit partial complementarity to their target mRNAs, primarily in the 3' untranslated region (UTR). * This partial complementarity usually leads to the inhibition of translation rather than direct cleavage of the mRNA. #### 2.2.2 siRNA vs. miRNA While both siRNAs and miRNAs are small RNA molecules processed by Dicer and integrated into RISC, they differ in their origin and primary mode of action: * **siRNAs:** Originate from exogenous or endogenous long dsRNAs and typically cause cleavage of perfectly matched target mRNAs. * **miRNAs:** Originate from endogenous genes (miRNA genes) and usually lead to translational repression through partial complementarity to target mRNAs. > **Tip:** While siRNAs predominantly cause mRNA cleavage and miRNAs cause translational repression, there can be overlap. Some siRNAs can inhibit translation, and some miRNAs can induce mRNA degradation, especially with near-perfect complementarity. #### 2.2.3 RNA interference (RNAi) vectors RNAi can be stably or transiently introduced into cells using various vector systems to achieve gene silencing. * **siRNA and shRNA delivery:** * **Chemically synthesized siRNAs:** These are synthetic RNA molecules that can be directly delivered into cells for transient knockdown. * **shRNA expression vectors:** These vectors allow for the continuous expression of short hairpin RNAs (shRNAs) within the cell. shRNAs are processed into siRNAs by Dicer. * **Plasmid-based vectors:** These plasmids contain a promoter (e.g., U6) that drives shRNA transcription *in vivo*. * **Transient transfection vectors (e.g., pSIREN-shuttle):** These vectors are designed for temporary expression and do not integrate into the host genome. They are suitable for short-term gene knockdown studies. * **Stable transfection vectors (e.g., pSIREN-RetroQ):** These vectors contain retroviral LTRs, enabling integration into the host genome for long-term, stable gene silencing. * **Viral vectors:** Lentiviral vectors or other viral systems can be engineered to express shRNAs, facilitating stable integration and long-term gene knockdown in a wider range of cell types, including non-dividing cells. * **Antisense Oligonucleotides (ASOs):** Single-stranded DNA or RNA molecules that bind to complementary mRNA sequences. * **Mechanism:** ASOs can block translation or modulate splicing, leading to protein downregulation. * **AntagoNATs:** These are antisense transcripts designed to block the action of endogenous miRNAs, leading to protein upregulation. > **Example:** Antisense-mediated exon skipping is a therapeutic strategy being explored for diseases like Duchenne muscular dystrophy. ASOs are designed to block specific splicing sites, causing the skipping of certain exons and potentially restoring a partially functional protein. ### 2.3 Other post-transcriptional regulatory mechanisms Besides RNAi, several other processes regulate gene expression after transcription: * **mRNA localization:** Directs where in the cytoplasm proteins are synthesized, ensuring localized protein function. * **Translational control:** Mechanisms that regulate the rate at which ribosomes translate mRNA into protein. * **mRNA surveillance (e.g., NMD - Nonsense-mediated decay):** Quality control pathways that detect and degrade aberrant or faulty mRNA molecules, preventing the production of non-functional or harmful proteins. * **Polyadenylation:** The addition of a poly(A) tail to mRNA, which can influence mRNA stability and translation efficiency. ### 2.4 Therapeutic applications of RNAi RNAi-based therapies hold significant promise for treating a wide range of diseases by specifically silencing disease-causing genes. * **Mechanism of action for therapeutic agents:** * **ASOs:** Can block mRNA translation or alter splicing patterns. * **siRNA/shRNA:** Induce the knockdown of target mRNA levels by promoting their degradation. * **Drug inhibitors:** Molecules that directly bind to and inhibit the function of specific proteins involved in gene expression pathways. * **Ribozymes:** Catalytically active oligonucleotides that can promote mRNA degradation. * **Development and delivery:** * RNAi therapeutics can be produced through chemical synthesis or *in vivo* transcription using expression vectors. * Delivery strategies are crucial for effective therapeutic outcomes and include chemical modification of RNA molecules, conjugation to targeting ligands, and encapsulation in nanoparticles or viral vectors. * **Clinical examples:** * **Duchenne muscular dystrophy:** Antisense oligonucleotides are being investigated to restore dystrophin protein expression by promoting exon skipping. * Other applications are being explored for genetic disorders, viral infections, and cancer. ### 2.5 Key Definitions * **miRNA (Micro RNA):** A small, non-coding RNA molecule (typically 21 nucleotides) involved in post-transcriptional regulation, usually by inhibiting translation. * **siRNA (Small interfering RNA):** A small, non-coding RNA molecule (typically 21 nucleotides) derived from longer dsRNA that mediates gene silencing by directing the cleavage of target mRNA. * **shRNA (Short hairpin RNA):** An artificial RNA molecule with a hairpin structure that is processed into siRNA within the cell. * **dsRNA (Double-stranded RNA):** RNA with two complementary strands. * **Dicer:** An enzyme that cleaves dsRNA into small RNA fragments (siRNAs and miRNAs). * **RISC (RNA-induced silencing complex):** A protein complex that incorporates small RNAs (siRNA or miRNA) to guide them to target mRNAs for silencing. * **ASO (Antisense Oligonucleotide):** A short, synthetic strand of DNA or RNA designed to bind to a specific mRNA sequence to inhibit translation or modulate splicing. * **AntagoNAT:** An antisense transcript that blocks the activity of endogenous miRNAs. * **pri-miRNA:** The initial precursor transcript of a miRNA in the nucleus. * **pre-miRNA:** The hairpin-shaped precursor of a miRNA, processed from pri-miRNA. * **Passenger strand:** The strand of an siRNA or miRNA duplex that is typically degraded or discarded by RISC. * **Guide strand:** The strand of an siRNA or miRNA duplex that is retained by RISC to direct it to target mRNA. * **Off-target effects:** Unintended silencing of genes other than the intended target, often due to partial complementarity of siRNAs/miRNAs to non-target mRNAs. * **On-target recognition and cleavage:** The precise binding and subsequent degradation of the intended target mRNA by the RNAi machinery. --- # Selection systems and cell culture media This section details strategies for identifying successfully gene-transferred cells and explores the various media formulations utilized for eukaryotic cell cultivation. ### 3.1 Selection systems for gene transfer Following gene transfer, it is crucial to identify and isolate cells that have stably integrated the introduced genetic material. This is typically achieved through the use of selection systems, which exploit specific cellular responses to selective agents or pressures. #### 3.1.1 Selection pressure Selection pressure is applied using agents that are toxic to cells unless they possess a specific resistance conferred by the introduced gene. * **Example: Methotrexate (Mtx) and Dihydrofolate Reductase (DHFR)** A common selection system involves using methotrexate (Mtx) as a selective agent. Methotrexate inhibits the enzyme dihydrofolate reductase (DHFR), which is essential for DNA synthesis and cell proliferation. If the gene of interest is co-transfected with a functional *DHFR* gene, cells expressing *DHFR* can survive in the presence of Mtx. Furthermore, if the *DHFR* gene is amplified, leading to increased DHFR expression, cells become more resistant to Mtx, allowing for the selection of cells with amplified gene copies of both *DHFR* and the co-transfected gene of interest. #### 3.1.2 Dominant selectable markers Dominant selection markers are genes that confer resistance to a selective agent, and their expression is independent of the host cell's endogenous genotype. This makes them versatile for use across different cell types. * **Neomycin resistance gene (NeoR)** The neomycin resistance gene encodes an enzyme, aminoglycoside phosphotransferase (APH), which inactivates the antibiotic G418. Cells expressing NeoR are therefore resistant to G418 and can be selected for in culture. #### 3.1.3 Complementary selection systems Complementary selection involves restoring a genetic defect in a cell by introducing a functional gene. * **Example: Thymidine Kinase (TK) and HAT medium** In this system, *TK-* deficient cells (cells lacking functional thymidine kinase) are co-transfected with a functional *TK* gene and the gene of interest. These cells are then cultured in HAT medium, which contains hypoxanthine, aminopterin, and thymine. Aminopterin blocks endogenous de novo nucleotide synthesis. Only cells that can synthesize nucleotides via the salvage pathway, which requires functional thymidine kinase, can survive and proliferate in HAT medium. Thus, successful co-transfection and expression of both the *TK* gene and the gene of interest are selected for. ### 3.2 Cell culture media Cell culture media are essential for maintaining the viability, growth, and function of eukaryotic cells in vitro. They provide the necessary nutrients, growth factors, and physical conditions for cell survival. #### 3.2.1 Common cell culture media formulations Several standard media formulations are widely used in cell culture: * **RPMI (Roswell Park Memorial Institute) medium:** Often used for suspension cell cultures. * **Opti-MEM (Optimized Minimal Essential Medium):** A reduced serum medium designed to improve transfection efficiency and reduce variability. * **EMEM (Eagle's Minimal Essential Medium):** A basal medium providing essential amino acids, vitamins, and salts. * **DMEM (Dulbecco's Modified Eagle Medium):** A widely used basal medium, often supplemented with higher concentrations of amino acids and vitamins than EMEM, and available with varying glucose concentrations. ### 3.3 Cell culture media and transfection The choice of cell culture medium can significantly impact the efficiency of gene transfer techniques, particularly transfection. Some media, like Opti-MEM, are specifically formulated to enhance transfection rates. > **Tip:** When performing transfections, it is often beneficial to switch to a reduced-serum medium like Opti-MEM for a period before, during, and after the transfection procedure to minimize interference from serum components with the transfection reagents and complexes. ### 3.4 Media considerations for selection When using selection systems, it is critical that the cell culture medium not only supports cell growth but also contains the necessary components for the selection process. For example, the selection agent (e.g., G418, methotrexate) must be added to the appropriate basal medium. #### 3.4.1 Selection in specific media * **HAT medium:** As described in the TK selection system, HAT medium is a specialized formulation essential for selecting cells that have successfully incorporated and are expressing the *TK* gene. ### 3.5 Plant cell culture media and transformation While the primary focus here is on animal cell culture, it's worth noting that plant cells also have specific media requirements. For transformation techniques in plants, such as those utilizing *Agrobacterium tumefaciens* or biolistics, appropriate plant growth media are crucial for the regeneration and growth of transformed cells into whole plants. These media typically contain macronutrients, micronutrients, vitamins, amino acids, and plant hormones. --- ## Common mistakes to avoid - Review all topics thoroughly before exams - Pay attention to formulas and key definitions - Practice with examples provided in each section - Don't memorize without understanding the underlying concepts

| Term | Definition | |------|------------| | Transfection | The process of introducing foreign nucleic acids (DNA or RNA) into eukaryotic cells. This can result in transient expression (temporary) or stable expression (permanent integration into the genome). | | Transduction | A method of gene transfer that utilizes viruses as vectors to deliver genetic material into host cells. This often leads to stable integration of the viral DNA into the host genome. | | Transformation | Historically, this term referred to the uptake of foreign DNA by bacterial cells. In broader contexts, it can also describe the genetic alteration of a cell, but in molecular biology, transfection is more commonly used for eukaryotes. | | MOI (Multiplicity of Infection) | A measure used in virology to quantify the number of infectious virus particles (virions) per cell when infecting a population of cells. It is calculated as the ratio of virus particles to target cells. | | siRNA (Small interfering RNA) | A type of small RNA molecule, typically 20-25 nucleotides in length, that plays a role in gene silencing through the RNA interference pathway. siRNAs are usually derived from longer double-stranded RNA precursors. | | miRNA (Micro RNA) | Small non-coding RNA molecules, approximately 21-23 nucleotides long, that regulate gene expression by binding to complementary sequences on messenger RNA (mRNA) molecules, typically leading to translational repression or mRNA degradation. | | RISC (RNA-induced silencing complex) | A multiprotein complex that mediates RNA interference (RNAi) and transcriptional gene silencing. The complex incorporates a small RNA (like siRNA or miRNA) and is guided to target nucleic acid sequences. | | shRNA (Small hairpin RNA) | A type of RNA molecule that forms a hairpin structure and can be processed into siRNA within the cell. shRNAs are often used experimentally to induce RNA interference and silence specific genes. | | Plasmid | A small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells and can also be found in some eukaryotes. In molecular biology, they are often used as vectors for gene cloning and expression. | | Viral Vector | A virus that has been genetically modified to deliver genetic material into cells. Viral vectors are widely used in gene therapy and molecular biology research for efficient gene transfer due to their natural ability to infect cells and integrate or express their genetic payload. | | Electroporation | A technique used to introduce foreign molecules, such as DNA or RNA, into cells by applying a brief electrical pulse. The electric field creates temporary pores in the cell membrane, allowing the genetic material to enter. | | Lipofection | A method of gene transfection that uses liposomes (lipid-based vesicles) to deliver nucleic acids into cells. The liposomes encapsulate the genetic material and fuse with the cell membrane, releasing their cargo into the cytoplasm. | | Exon Skipping | A type of RNA splicing abnormality where an exon is omitted from the mature messenger RNA (mRNA) during the splicing process. This can be therapeutically induced, for example, using antisense oligonucleotides to correct mutations in genetic disorders. | | Poly-adenylation | The addition of a tail of multiple adenine nucleotides (poly-A tail) to the 3' end of a messenger RNA (mRNA) molecule. This process is crucial for mRNA stability, export from the nucleus, and translation efficiency. | | NMD (Nonsense-Mediated Decay) | A surveillance pathway in eukaryotic cells that degrades aberrant messenger RNAs (mRNAs) containing premature stop codons. This mechanism helps to prevent the production of truncated and potentially harmful proteins. | | Ti-plasmid | A plasmid found in the bacterium *Agrobacterium tumefaciens* that is capable of transferring a segment of its DNA (T-DNA) into the plant genome, causing crown gall disease. It is widely used as a vector for genetic engineering in plants. |