Molecular Cloning and Transfection Methods

These flashcards cover key concepts in molecular cloning and transfection methods as discussed in the lecture.

What are the main steps in bioprocess development for gene therapy?

Gene for Therapeutic Protein, Transfection, Host cell, Cell Pool, Selection, Candidate clones, Small Scale studies, Master Cell Culture, Expanded Manufacturing.

What is the goal of transfection/transformation in mammalian cells?

To deliver a therapeutic gene (transgene) to selected cells for gene expression.

What is the difference between transient and stable transfection?

Transient transfection does not integrate foreign DNA into the genome and results in temporary gene expression, while stable transfection integrates foreign DNA into the genome and results in permanent gene expression.

What is a plasmid?

A plasmid is a circular (mostly) DNA that can integrate fragments from a few base pairs up to 10kb DNA and is derived from bacteria.

What are the methods for gene transfer into mammalian cells?

Direct transfer of plasmid DNA or infection with a virus carrying the recombinant gene of interest.

What is lipofection?

Lipofection involves synthesizing liposomes that encapsulate nucleic acids and facilitate their entry into cells by endocytosis.

What is the advantage of using microinjection for gene delivery?

Microinjection allows for targeted gene delivery into specific cells but is limited by the number of cells that can be effectively transfected.

What is optoporation?

Optoporation uses femtosecond laser pulses to create transient perforations in a cell membrane to insert genetic material.

What are the advantages of electroporation?

Electroporation is a highly efficient technique that creates transient pores in the cellular membrane allowing DNA entry, but can cause cell damage.

What is the primary purpose of a selection marker in viral transduction?

To select for cells that have been successfully transduced with the virus.

What is the difference between lentivirus and adenovirus in terms of their genetic expression?

Lentivirus provides stable expression by integrating its RNA into the host genome, while adenovirus allows for transient expression without integration.

How does the Dihydrofolate Reductase (DHFR) system work in mammalian cell screening?

Cells with a high DHFR expression survive in the presence of a DHFR inhibitor, indicating successful transfection.

Describe the blue/white screening method in bacterial cell screening.

In blue/white screening, bacteria carrying a plasmid with an inserted gene turns white, while those without the insert remain blue due to active β-galactosidase.

What is the role of the glutamine synthetase (GS) system in mammalian cell selection?

GS is used as a selection marker to ensure that only cells with the correct gene are amplified in culture.

What is the CYTO-MINE technology used for?

CYTO-MINE technology is used to analyze individual cells or pools of cells quickly using picodroplets.

What are the challenges associated with transfection technology?

Difficult cell types, low efficiency and toxicity issues, and the need for safe/effective in vivo delivery methods.

How do cells with and without plasmid respond to antibiotic selection during bacterial transgenic cell screening?

Cells with the plasmid will survive and grow on antibiotic plates, while those without will die.