This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
• Many claims of superior transfection efficiency are made by companies who sell reagents for transfection– Caveat emptor, one San Diego company uses a
competitor’s product in house instead of the reagent they promote.
– one of the largest profit margin items in the industry• unless you own stock in a company selling the
• W. Szybalski (a very famous microbiologist) decided to set up a system whereby mammalian cells could be induced to take up DNA, much like bacteria - first successful report in 1962.
– To maximize success he also developed the HAT selection method.
– By analogy to bacterial transformation, it was discovered that successful DNA transfer was dependent on the formation of a co-precipitate of DNA with calcium phosphate
• after the method was well understood in 1973, it became widely used
• Graham and van der Eb (1973) Virology 52, 456-467 is the classic reference.
• Chen and Okayama (1987) Mol Cell Biol 7, 2745-52 (very high efficiency variant)
• General principle is to form a precipitate of DNA that can be taken up by endocytosis
– Mix DNA, in phosphate buffer with CaCl2 at precise pH and an insoluble CaPO4 precipitate forms
• precision in pH is critical, alterations of as little as 0.01 pH units affect efficiency
• leave on cells several hours to overnight• wash ppt off and add fresh medium
– OR add DNA and buffer to cells at low (3%) CO2.• Ppt forms automatically over time
• advantages– very simple– very inexpensive– extensive literature– works for most cell types
• disadvantages– adherent cells only– some touch and experience required to get good
precipitates– not particularly efficient in many cell types– many cells do not like adherent precipitate– difficult to automate or perform as a high throughput
• produce unilamellar liposomes and allow DNA to interact with them. Liposomes can be produced by:– sonication– extrusion through a small pore membrane– dilution into aqueous medium
• mix with cells and allow to interact• for a long time it was assumed that liposomes mediate fusion
with cell membranes. However endocytosis is now known to be the mechanism
• various formulations– cationic lipids only, e.g. DOTAP– mixture of cationic and neutral lipids, e.g. lipofectin
– very simple to perform and optimize - anyone can do it.– easy to automate, high throughput– reliable and reproducible– stable and transient assays work well– works well with many cell types and in vivo
• principle is that brief, strong electrical pulse creates transient pores in the cell membrane that allows exchange of molecules
• cells and DNA are placed into a cuvette between two plates.
– High DC voltage (500+ V) applied as a pulse• square wave form appears to work better than
exponential decay (best for bacteria)• possible optimizations are voltage, pulse length,
wave form.– Some experimentation with RF (radio frequency)
pulses suggests greater efficiency• but apparatus is not readily available
• advantages– very efficient when it works– quite effective at making stable transfectants (e.g. ES
cells)• disadvantages
– only works well for cells in suspension• devices for transfecting adherent cells do not
work very well and are cumbersome to clean– kills cells very effectively– expensive equipment and cuvettes– extensive optimization– very sensitive to salt concentrations
• infection is absolutely the highest efficiency method possible– 100% infection is routine
• DNA to be expressed is cloned into a virus that can infect your favorite cell type - two general types of virus utilized– retroviruses (RNA viruses), e.g. HIV
• tend to integrate• can be insertional mutagens!• Relatively small sized insert• narrow host range
– large DNA viruses (adenovirus, vaccinia)• extrachromosomal replication• tend to have broad host specificity• tend to be lytic• large inserts are possible
• many viral genes are not required for infective virions– nonessential genes are removed, thus allowing the virus
to accommodate foreign DNA.– Most such viruses requires a packaging strain to get
infective virus particles• primarily for biosafety
• field is primarily driven by gene therapy applications– most current information found in gene therapy literature– Pfeifer and Verma (2001) Gene Therapy: promises and
problems Ann. Rev. Genomics Hum. Genet. 2, 177-211
• in vivo expression screening– microinject pools of mRNA generated from
libraries and evaluate function– various channels, receptors and transporters
identified this way• protein expression system• electrophysiology
– advantages• long term expression of injected materials• cells do not divide• transcription is possible• apparatus is relatively inexpensive• easy to collect and store oocytes• unhurried injections
– disadvantages• cells do not divide• not a developing system, limited questions• nuclear and cytoplasmic injections may be
required– e.g. reporter gene must be put in nucleus,
• what would we like to know about a founder line? (contd)– is transgene expression as predicted?
• If the transgene is under the control of a tissue-specific promoter (e.g. its own), is it expressed in the correct tissue at the correct time in development?
– Tissue Northern blots– in situ hybridization
• If the transgene is expressed from a ubiquitous promoter, is it expressed ubiquitously?
• Applications– Transgenesis is a gain of function method
• doesn’t speak to necessity of a gene, unless a mutation is being rescued
– rescue of a mutation– promoter analysis
• identify temporal or spatial requirements for expression
• verify function of suspected enhancer elements– create models for dominant forms of human diseases– identify effects of misexpression
• particularly with genes showing temporally or spatially restricted expression, e.g. Hox genes
• advantages of transgenic technology– analysis is performed in vivo
• best test for gene regulation– much less difficult than targeted disruption– relatively high efficiency compared with targeting
• disadvantages– gain of function– no ability to target integration site– no control over copy number– injected DNA must contain all regulatory elements– can’t study transgenes with dominant lethal phenotypes
• Targeted disruption of genes is very desirable, wave of the future
– great to understand function of newly identified genes from genome projects
• produce a mutation and evaluate the requirements for your gene of interest
– good to create mouse models for human diseases• knockout the same gene disrupted in a human and
may be able to understand disease better and develop efficacious treatments
• excellent recent review is Müller (1999) Mechanisms of Development 82, 3-21.
• enabling technology is embryonic stem (ES) cells– these can be cultured but retain the ability to colonize
the germ line– essential for transmission of engineered mutations– derived from inner cell mass of blastula stage embryos– grown on lethally irradiated “feeder” cells which help
to mimic the in vivo condition• essential for maintaining phenotype of cells
• Applications (contd)– marking gene with reporter, enabling whole mount
detection of expression pattern (knock-in)• advantages
– can generate a true loss-of-function alleles– precise control over integration sites– prescreening of ES cells for phenotypes possible– can also “knock in” genes
• disadvantages– not trivial to set up– may not be possible to study dominant lethal
phenotypes– non-specific embryonic lethality is common (~50%)– difficulties related to selection cassette