Lecture 4: Manipulating the genome
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Questions and Answers

What is a notable advantage of miRNA transfection when expressed as lentiviral vectors (LV)?

  • It provides high efficiency when combined with OKSM. (correct)
  • It is completely free of any cellular footprints.
  • It has been extensively proven in various cell types.
  • It integrates into the genome effectively.

Which of the following is a disadvantage of using mRNA transfection?

  • The synthesis of modified RNA is technically challenging. (correct)
  • It introduces a footprint into the cellular genome.
  • It is low in efficiency compared to other methods.
  • There are numerous published studies on its effectiveness.

Which statement is true regarding the efficiency of non-viral methods as compared to viral methods?

  • Viral methods introduce immediate activity without genetic modification.
  • Efficiency of non-viral methods is often questionable. (correct)
  • Viral methods have lower activation of exogenous silencing mechanisms.
  • Non-viral methods generally have significantly higher efficiency.

What is one of the major limitations of protein transfection?

<p>It involves the direct introduction of reprogramming proteins. (A)</p> Signup and view all the answers

What challenge is associated with the synthesis of modified RNA in mRNA transfection?

<p>It has an unacceptably long transfection duration. (B)</p> Signup and view all the answers

Which of the following reprogramming factor combinations includes c-Myc?

<p>OSNLKM (A), OSKM (C)</p> Signup and view all the answers

What is the purpose of introducing reprogramming factors into somatic cells?

<p>To trigger genetic and epigenetic changes (D)</p> Signup and view all the answers

Which type of cells can be reprogrammed to pluripotent states according to the content?

<p>Various somatic cell types (B)</p> Signup and view all the answers

What stage follows the introduction of reprogramming factors in the reprogramming process?

<p>Reprogramming (A)</p> Signup and view all the answers

Which of the following best describes the Yamanaka factors?

<p>They consist of Oct4, SOX2, Klf4, and c-Myc (B)</p> Signup and view all the answers

What is the ultimate goal of selecting individual cell clones during the reprogramming process?

<p>To ensure robust pluripotency and growth characteristics (A)</p> Signup and view all the answers

Which transcription factors are omitted in the reprogramming combination OSK?

<p>c-Myc (B)</p> Signup and view all the answers

How does the reprogramming process impact the identity of the cells?

<p>It enables them to become pluripotent (A)</p> Signup and view all the answers

What is a significant disadvantage of lentiviral transduction in reprogramming?

<p>Genomic integration can cause insertional mutagenesis (C)</p> Signup and view all the answers

What advantage does a polycistronic lentiviral vector provide in gene expression?

<p>Ensures balanced expression of multiple genes (C)</p> Signup and view all the answers

Which gene expression pattern occurs during the reprogramming process?

<p>Somatic gene expression decreases as pluripotency-related gene expression increases (C)</p> Signup and view all the answers

What are the main components delivered by lentiviral transduction?

<p>Reprogramming factors into target cells (B)</p> Signup and view all the answers

What is a key characteristic of excisable polycistronic vectors in reprogramming?

<p>They include multiple reprogramming factors in a single vector. (D)</p> Signup and view all the answers

Which of the following is NOT an advantage of using viral reprogramming methods?

<p>High efficiency for all reprogramming factors (C)</p> Signup and view all the answers

How does RNA-mediated reprogramming primarily differ from viral-mediated methods?

<p>RNA methods do not integrate into the genome. (A)</p> Signup and view all the answers

What defines the use of TetO in reprogramming?

<p>It provides flexibility in gene expression timing. (B)</p> Signup and view all the answers

Which of the following factors is known to enhance the efficiency of achieving iPSCs?

<p>Balanced stoichiometry of reprogramming factors (D)</p> Signup and view all the answers

What is one significant advantage of using excisable markers in reprogramming?

<p>They allow for efficient factor removal after reprogramming. (D)</p> Signup and view all the answers

Which of the following is NOT a disadvantage associated with Adenovirus transduction?

<p>It can infect non-dividing cells. (A)</p> Signup and view all the answers

What is a primary disadvantage of Sendai virus transduction?

<p>It requires culture at elevated temperatures. (C)</p> Signup and view all the answers

Which of the following methods is used to insert reprogramming factors into the genome?

<p>PiggyBac transposon transfection (A)</p> Signup and view all the answers

Which statement about non-viral methods of transfection is accurate?

<p>They include methods such as siRNA and protein transfection. (A)</p> Signup and view all the answers

What is a characteristic feature of the Sendai virus used in transduction?

<p>It produces high levels of protein. (B)</p> Signup and view all the answers

Which disadvantage is common to both Sendai virus and Adenovirus transduction methods?

<p>Both are difficult to produce. (C)</p> Signup and view all the answers

What is a common characteristic of the reprogramming methods discussed?

<p>Many methods have difficulties in production. (A)</p> Signup and view all the answers

How do excisable markers differ from non-excisable markers in genetic reprogramming?

<p>Excisable markers allow removal after use to reduce disruptions. (B)</p> Signup and view all the answers

What is a potential negative effect of using LTR fragments in genetic reprogramming?

<p>They can interfere with host genome stability. (D)</p> Signup and view all the answers

What is a key advantage of using transposons in genetic manipulation?

<p>They offer good efficiency and are non-viral. (C)</p> Signup and view all the answers

Which disadvantage is associated with minicircle vectors?

<p>They can only be used on transformed cell lines. (D)</p> Signup and view all the answers

What is a drawback of using episomal plasmids for transfection?

<p>They are often difficult to introduce into certain primary cell types. (A)</p> Signup and view all the answers

In contrast to other methods, which statement best describes the role of minicircle vectors?

<p>They are derivatives that minimize DNA sequence for specific factors. (D)</p> Signup and view all the answers

How do transposons differ from viral vectors in genetic manipulation?

<p>Transposons are non-viral and have a minimal genetic footprint. (C)</p> Signup and view all the answers

What is a significant limitation when working with transposons?

<p>They require an additional excision step and selection. (D)</p> Signup and view all the answers

What is the essential characteristic of episomal plasmids in terms of longevity within cells?

<p>They exist as extrachromosomal elements that are often lost. (D)</p> Signup and view all the answers

What is a primary challenge of using episomal plasmids for certain cell types?

<p>They often necessitate expensively complex machinery for efficient delivery. (C)</p> Signup and view all the answers

Which factor limits the use of transposons in genetic engineering?

<p>They can integrate into the host genome. (A)</p> Signup and view all the answers

What aspect of minicircle vectors makes them advantageous for transient expression?

<p>They minimize the necessary genetic sequence. (C)</p> Signup and view all the answers

Flashcards

Pluripotency

The ability of a cell to develop into any cell type in the body.

Pluripotent cells

Cells that are not specialized and can develop into any cell type in the body.

Reprogramming

The process of converting a specialized cell (like a skin cell) into a pluripotent cell.

Reprogramming factors

Factors (proteins) that trigger genetic and epigenetic changes to reprogram a cell.

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Yamanaka factors (OSKM)

A combination of reprogramming factors commonly used to create induced pluripotent stem cells (iPSCs).

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Somatic cell reprogramming

The process of generating induced pluripotent stem cells (iPSCs) from somatic cells.

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Variability in somatic cell reprogramming

The variability seen in the efficiency of generating iPSCs from different cell types or using different reprogramming methods.

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iPSC clones

Individual cells that have obtained pluripotency and are capable of self-renewal.

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Pluripotency Gene Expression in Reprogramming

Genes responsible for pluripotency (ability to become any cell type) are expressed more during reprogramming.

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Lentiviral Transduction

A method using lentiviruses to deliver reprogramming factors into target cells.

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Lentiviral Transduction: Infecting Non-dividing Cells

The ability of lentiviral transduction to infect non-dividing cells.

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Genomic Integration and Insertional Mutagenesis

A disadvantage of lentiviral transduction: the insertion of viral DNA into the target cell's genome can lead to mutations.

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Polycistronic Lentiviral Vector

A method using a single vector containing multiple reprogramming factors for efficient delivery.

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Polycistronic Vectors: Balanced Expression

The advantage of a polycistronic vector: balanced expression of multiple genes, ensuring proper production of all reprogramming proteins.

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Inducible Polycistronic Vectors

A type of polycistronic vector allowing for controlled timing of gene expression.

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Size Constraints of Viral Packaging

A disadvantage of lentiviral transduction: size constraints limit the number of factors that can be packaged into a single virus.

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Excisable Polycistronic Vectors

Vectors containing reprogramming factors in a polycistronic format where select, extraneous elements can be removed.

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Excisable Marker

A gene editing technique that involves removing reprogramming factors after they've been inserted into the genome.

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Adenovirus Transduction

A method for delivering reprogramming factors using adenoviral vectors. The virus doesn't integrate into the host genome, resulting in temporary expression of the factors.

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Sendai Virus Transduction

A method for delivering reprogramming factors using Sendai viruses. These viruses don't integrate into the host genome, ensuring transient expression.

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Piggybac Transposon Transfection

A reprogramming method that uses the PiggyBac transposon system to insert reprogramming factors into the genome.

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Viral Reprogramming

A method of reprogramming that uses a specific type of virus to insert reprogramming factors into a cell.

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Non-Integrating Virus

A method that uses a virus that does not integrate into the host genome, resulting in transient expression of reprogramming factors.

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Non-Viral Reprogramming

A method of reprogramming where the reprogramming factors are delivered to the cell without using a virus.

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LTR Fragments

A concern with using excisable markers for reprogramming. Small fragments of the marker can remain in the genome.

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Non-Pathogenic

An advantage of Sendai virus transduction. The virus is non-pathogenic, meaning it does not cause disease.

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Viral RNA Dilution

A disadvantage of Sendai virus transduction. The virus takes several passages to fully dilute out, requiring careful handling.

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mRNA transfection

Introducing reprogramming factors as mRNA into cells. This mRNA gets translated into proteins without integrating into the cell's DNA.

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Protein transfection

Directly introducing the reprogramming proteins into cells, bypassing the need for gene delivery or DNA integration.

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Polycistronic Vectors

A method using a single vector containing multiple reprogramming factors for efficient delivery. This helps ensure a balanced production of all necessary proteins.

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Transposon-mediated reprogramming

A type of gene editing method that uses transposons to deliver reprogramming factors. These transposons are later removed, leaving minimal genetic changes.

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Viral delivery

A viral delivery method used in genome editing where viruses are engineered to carry reprogramming factors into cells.

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Transposon-based reprogramming: Advantages

A gene editing method using transposons that offers advantages like non-viral delivery, inducible expression, and excisable reprogramming factors.

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Transposon-based reprogramming: Disadvantages

A gene editing method using transposons that comes with drawbacks like the need for a second step to remove the transposon and labor-intensive procedures.

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Episomal plasmid transfection

A gene editing method using extrachromosomal plasmids to deliver reprogramming factors. These plasmids eventually disappear from the cells.

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Episomal plasmid transfection: Advantages

A gene editing method that avoids integration into the genome and leaves no footprint.

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Episomal plasmid transfection: Disadvantages

A gene editing method using episomal plasmids that can be inefficient and challenging to apply to certain cell types.

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Minicircle vectors

A gene editing method that uses small, circular DNA molecules (minicircles) to deliver reprogramming factors. Minicircles are non-integrating and often used for temporary expression.

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Genome editing

A type of genetic engineering technique used to change the genetic makeup of cells, often with the goal of creating induced pluripotent stem cells.

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Study Notes

Lecture 4: Manipulating the Genome

  • Learning Objective (LO): Cells from diverse origins can be reprogrammed to a pluripotent state. Variability in somatic cell reprogramming often stems from donor cell type and reprogramming method.

Many Somatic Cell Types Suitable for Reprogramming

  • Many somatic cell types are suitable for reprogramming (e.g., fibroblasts from mice or humans, blood cells, renal tubular cells, keratinocytes).
  • Yamanaka factors (Oct4, SOX2, Klf4, c-Myc) are commonly used, and are often combined in varying selections depending on the cell type of origin.
  • Thomson factors (Oct4, Sox2, Nanog, Lin28) can also be combined as alternative combinations.
  • A specific combination of factors may be more effective for reprogramming some types of cells.

Reprogramming is a Staged Process

  • Reprogramming is a cellular process.
  • Somatic cell reprogramming typically involves isolating cells, introducing reprogramming factors, selecting clones with robust pluripotency characteristics, expanding, and characterizing the induced pluripotent stem cells (iPSCs).
  • Confirmation involves checking for, and confirming molecular markers of pluripotency.

Preparing Somatic Cells and Reprogramming

  • Somatic cells are isolated from a donor or patient.
  • Specific transcription factors (e.g., OSKM) are introduced using methods like viral vectors.
  • These introduced factors induce changes in gene expression and epigenetic modifications, ultimately resetting the cell's identity, allowing it to become pluripotent.
  • Clones are screened to select those with robust pluripotency and characteristics.
  • iPSCs are then expanded and characterized to ensure quality.

Reprogramming is a Molecular Process

  • Somatic gene expression initially high and decreases as reprogramming occurs; while pluripotency-related gene expression increases over time.

Viral, RNA, and Protein-Mediated Reprogramming

  • Different methods for introducing reprogramming factors include viral (lentiviral, adenoviral, Sendai virus) methods, RNA, and protein-mediated approaches.
    • Viral: Lentiviral transduction uses lentivirus particles to deliver reprogramming factors to cells; adenoviral vectors introduce factors without integrating into the genome (a transient effect); and Sendai viruses are likewise non-integrating. Considerations for each vector method include efficiency of transduction, possibility of insertional mutagenesis (viral integration into the genome), and difficulties in production.
    • RNA: techniques involve using mRNA encoding reprogramming factors to introduce the factors into the cells.
    • Protein: introducing proteins directly into cells to activate the reprogramming process.
  • Specific Advantages and Disadvantages exist for each.

Excisable Polycistronic Vectors

  • These vectors contain reprogramming factors in a polycistronic format and a selection marker.
  • The marker is excised after reprogramming, preventing interfering effects.

Reprogramming Methods (viral/RNA/protein)

  • Inducible varieties: gene expression can be regulated using TetO or more flexible systems enabling precise timing of factor introduction and expression.
  • Adenovirus Transduction: Factors are delivered using adenoviral vectors. Transient expression. Can infect non-dividing cells.
  • Sendai Virus Transduction: Used to deliver factors, but does not integrate into the genome, a non-integrating approach.

Non-Viral Methods

  • PiggyBac transposon transfection: uses the PiggyBac transposon system to insert reprogramming factors into the genome. Transposons can be excised.
  • Episomal plasmids transfection involves introducing plasmids carrying reprogramming factors. Plasmids often exist as episomes and will not integrate into the genome.
  • Minicircle vectors: are non-integrating plasmids with minimal DNA and reprogramming factors. Used for transient expression.
  • mRNA transfection: introduces RNA molecules encoding reprogramming factors, to be translated into proteins without integration.
  • miRNA transfection enhances reprogramming efficiencies but has some limitation in the cell and specific types, like human fibroblasts.

Protein Transfection

  • Method of introducing reprogramming proteins directly into cells.
  • Advantages: immediate activation, non-integrating.
  • Disadvantages: requires producing biologically active proteins that can cross cell membranes. Low efficiency, fewer published studies.

Validating Genetic Disease Phenotypes in iPSCs

  • Using iPSCs, researchers evaluate the correlation between specific genetic mutations and corresponding phenotypes. A key technique to study the impact of mutations on cell functions involves either introducing mutations or correcting existing mutations using gene-editing techniques.

Regulating Genome Expression using CRISPR/Cas9 Technology

  • Research now uses CRISPR/Cas9 technology to regulate genomic expression.

Taking the Next Step in Differentiation

  • Pluripotent cells can be induced to differentiate, taking a step into a specific cell type.

LO: Taking the Next Step in Differentiation

  • Pluripotency can be induced in stem cells and then induced again, further into different pathways of differentiation.

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Lecture 4: Manipulating the Genome - PDF

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Explore the complexities of somatic cell reprogramming in this quiz. Learn about the different somatic cell types that can be reprogrammed and the specific factors used in the process. Understand the stages involved in transforming these cells to a pluripotent state.

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