Lecture 17

Question 1

define transfection and explain experimental goals of this procedure and how it is performed practically

Answer 1

transfection: method by which experimental DNA or RNA is put into a cultured mammalian cell. usually recombinant DNA (plasmids) or RNA (siRNAs, miRNAs)
goals: test if DNA can be expressed/knocked down, where are recombinant proteins localized, what is protein function, how does a particular modification affect the gene function, function/effect of miRNAs, gene editing via CRISPR

Question 2

explain the difference between a transient and a stable transfection

Answer 2

transient transfection: foreign DNA is not inserted into the nuclear genome (episcopal). the DNA is lost or diluted during cell divisions
stable transfection: this is the goal. transfected gene integrates in the genome and persists in cells, very hard to accomplish. procedure involves using a selective marker for advantage, still very few will be randomly integrated. add the selective toxin and then clonal growth of the transfected cells

Question 3

describe calcium phosphate method steps, pros and cons

Answer 3

chemical transfection, THE classical method.

1. separate solutions containing DNA in phosphate buffer and calcium ions (salt)
2. Ca and phosphate combine and precipitate as Ca3(PO4)2
3. DNA is trapped in or attached to precipitates
4. cells take up DNA containing precipitates by endocytosis
5. wash, incubate, assay, etc

Pros: easy, inexpensive, can be transient or select stable
Cons: doesn’t work for all cell types (not primary cells), efficiency is low to moderate <10%, needs a lot of DNA, requires optimization, hard to reproduce, Cytotoxic! disrupts cell membrane and leads to cell death

Question 4

describe DEAE dextran method steps, pros and cons

Answer 4

chemical transfection, uses polycationic polymer

1. solutions containing DEAE dextran and DNA are mixed (electrostatic interactions)
2. add mixture to cells, complex adheres to cells
3. induce uptake (osmotic shock, DMSO, etc)
4. wash, incubate, assay etc

pros: easy, reproducible, inexpensive, transient
cons: not for all cell types, low efficiency in primary cells, rehires reduced serum for transfection, not for stable lines, Cytotoxic!

Question 5

describe liposome transfection steps, pros and cons

Answer 5

chemical transfection, most widely used non-viral

1. dilute DNA and transfection reagent
2. combine them, complexes form (neg charge DNA and pos charge lipids)
3. add to cells, complex enters via endocytosis
4. incubate, assay, etc

Pros: easy, reproducible, transient and stable, needs less DNA, less cytotoxic, suitable for RNAs and DNAs, optimized reagents available for specific cell types, in vivo delivery possible (vaccines)
Cons: expensive, some cytotoxicity

Question 6

describe microinjection, pros and cons

Answer 6

physical method. microinjection uses finely drawn capillaries to deliver DNA directly into the cell. application mostly (but not limited to) reproductive biology and animal transfenesis (oocytes)

pros: effective for primary cells, DNA less modified, precise control of DNA amount
cons: expensive equipment, slow (inject one at a time), requires significant operator skill

Question 7

describe electroporation, pros and cons

Answer 7

physical method. uses electric field (high voltage pulse) to open up pores in membrane to allow DNA to enter. use buffers with less salt, critical parameters of maximum voltage of the shock and duration of the pulse

pros: easy, reproducible, high efficiency for transient and stable, effective for suspension cells and some primary
cons: very damaging, usually kills 70-80%, requires more DNA and cells, requires optimization, expensive equipment

Question 8

describe nucleofection

Answer 8

electroporation + lipofection. optimized electroporation parameters and cell type specific reagents and devices
good for primary cells, high efficiencies, very expensive

Question 9

describe viral gene transfer (TRANSDUCTION) pros and cons

Answer 9

uses receptor mediated uptake which can be specific to cell types if you select specific receptors. common vectors are retroviruses, AAV, adenoviruses, or synthetic vectors

pros: very efficient, cell type specificity, in vivo possible (gene therapy)
cons: production is costly and labor intensive (after first making it is much easier and quicker to use), limited capacity, biosafety risk if made for human cells