Bio Sci 145 A Lecture 11 2122002 Transgenic

Bio. Sci 145 A Lecture 11 - 2/12/2002 Transgenic technology and its implications • Topics we will cover today – how to get the DNA into cultured cells. • chemical transfection of cultured cells • cell fusion (won’t discuss) • liposome-mediated transfer • cationic dendrimers • virus mediated • electroporation – how to get DNA into embryos • biolistic gene transfer • microinjection • electrotransformation • viral infection – transgenic technology • standard transgenesis • gene targeting – Dr. La Morte will discuss single cell microinjection techniques Bio. Sci 145 A lecture 11 page 1 ©copyright Bruce Blumberg 2000. All rights reserved

How to get DNA into cells - introduction • • General terminology – transformation refers to the uptake of foreign DNA, e. g. plasmid transformation of bacteria. – Transfection, strictly speaking, refers to the transfer of viral DNA – when referring to animal cells, we tend to use the term transfection to distinguish the transfer of DNA from the “transformation” of a cancer cell. Transfection efficiency varies greatly from one type of cell line to another using any method. – Must usually test several methods to determine which one works best for your cells and hands. Stable vs. transient transfections is also relevant. – Supercoiled plasmids are best for transient transfections, linear best for stable transfections – stable transfectants usually have single integration site with multiple copies integrated – transient transfectants may replicate extrachromosomally. Observation is that cells that take up any DNA take up all DNA – e. g. if cells take up one type of plasmid from the surroundings, they will take up all types – enables co-transfection, introduction of multiple plasmids/cell – this is a fundamental and indispensable tool Bio. Sci 145 A lecture 11 page 2 ©copyright Bruce Blumberg 2000. All rights reserved

How to get DNA into cells - introduction (contd) • 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 reagents, make your own whenever possible Bio. Sci 145 A lecture 11 page 3 ©copyright Bruce Blumberg 2000. All rights reserved

Chemical transfection - Ca 3(PO 4)2 • • 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 Ca. Cl 2 at precise p. H and an insoluble Ca. PO 4 precipitate forms • precision in p. H is critical, alterations of as little as 0. 01 p. H 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%) CO 2. • Ppt forms automatically over time Bio. Sci 145 A lecture 11 page 4 ©copyright Bruce Blumberg 2000. All rights reserved

Chemical transfection - Ca 3(PO 4)2 (contd. ) • • 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 method Bio. Sci 145 A lecture 11 page 5 ©copyright Bruce Blumberg 2000. All rights reserved

Chemical transfection - DEAE dextran • • • diethylaminoethyl (DEAE) modified dextran is a positively charged polymer – many other charged polymers have been used with varying degrees of success and reproducibility • PEI - polyethyleneimine • poly-L-lysine • roll your own DNA adheres to the polymer and remains soluble by some unknown means, the complex interacts with the cells and is taken up by endocytosis advantages – may work in cells that are refractory to other methods – gentle, not very toxic to cells – works for cells in suspension disadvantages – doesn’t work well in many cell types – doesn’t work well for stable transfectants – unclear mechanism of action makes optimization troublesome – moderately expensive – low throughput Bio. Sci 145 A lecture 11 page 6 ©copyright Bruce Blumberg 2000. All rights reserved

Lipofection - liposome mediated transfection • • • 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 (DOTMA: DOPE) – phospholipids – cholesterol-related lipids all work to some degree advantages – 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 • adherent and nonadherent Bio. Sci 145 A lecture 11 page 7 ©copyright Bruce Blumberg 2000. All rights reserved

Lipofection - liposome mediated transfection (contd) • disadvantages – many formulations require use of serum free, or serum reduced medium for good efficiency • all types that use neutral lipids – some formulations are unstable to oxygen • DOTAP and other unsaturated lipids – variable toxicity necessitates careful optimization for many types (e. g. Lipofectin) – VERY expensive to buy (but almost free to make) – for example • BMB-Roche sells 2 mg of DOTAP transfection reagent for $285. This is enough for ~6 96 -well plates ($48/plate) – 1 gram = $142, 500 • pure DOTAP costs ~$400/gram from Avanti Polar Lipids. Time and material to make liposomes in vials about doubles this cost. About $0. 20/96 -well plate – Manufacturers lie quite a bit about the performance of their reagents due to the profit margins • many do not work well, others not at all Bio. Sci 145 A lecture 11 page 8 ©copyright Bruce Blumberg 2000. All rights reserved

Cationic dendrimer mediated transfection • • • polycationic polymers of various densities and patterns (e. g. Superfect) interact with DNA to form complexes these interact with cells and are taken up by endocytosis advantages – may be more efficient than liposomes – stable and easy to use – low toxicity – automation friendly, high throughput – suspension or adherent cells disadvantages – expensive – not readily possible to synthesize Bio. Sci 145 A lecture 11 page 9 ©copyright Bruce Blumberg 2000. All rights reserved

Electroporation - electricity driven transfection • • 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 Bio. Sci 145 A lecture 11 page 10 ©copyright Bruce Blumberg 2000. All rights reserved

Viral infection • • 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 Bio. Sci 145 A lecture 11 page 11 ©copyright Bruce Blumberg 2000. All rights reserved

Viral infection (contd) • • advantages – efficiency – simplicity of infection disadvantages – not really feasible to introduce multiple constructs per cell. Best for introducing a single cloned gene that is to be expressed highly – at least P 2 containment required for most viruses • lots of hoops to jump through with institutional review boards (IRB) • viral transfer of regulatory genes, or oncogenes is inherently dangerous and should be carefully monitored • not so many old virologists – host range specificity may not be adequate – many viruses are lytic – need for packaging cell lines Bio. Sci 145 A lecture 11 page 12 ©copyright Bruce Blumberg 2000. All rights reserved

How to get DNA into cells - summary • • • common feature of nearly all transfection methods is to form dense DNA complexes of small, uniform size – 75 -100 nm seems best how the complex is made may not matter much, many variations are possible (thousands of papers) – size uniformity of particles is strongly related to efficiency of transfection needs to be optimized for the type of cells and requirements of each experiment which method is the best one for me? – What is working in the lab or surrounding labs? • Troubleshooting is rate limiting step in science – liposomes and cationic dendrimers generally the best • fast • reproducible • broad applicability – if cost is a concern, either make your own liposomes or use calcium phosphate – electroporation and viral infection have important utility but restricted applicability • electroporation is great for cells in suspension • viral infection is great for a single gene single cell microinjection is now feasible (Dr. La Morte) – throughput is low – uniform delivery ensures reproducibility Bio. Sci 145 A lecture 11 page 13 ©copyright Bruce Blumberg 2000. All rights reserved

How to get DNA into embryos (other than mouse) • • • Why would we want to do this anyway? – Determine function of identified genes – develop animal models for various diseases – confer desirable property Choice of method depends on model system, developmental stage and outcome desired – early embryos • if cells are large then direct microinjection is possible (e. g. Xenopus and zebrafish) • otherwise use methods below – later embryos, cells are too small for direct microinjection • biolistic gene transfer • electrotransformation • viral infection • liposome-mediated transfer transgenic techniques - germline transmission – must be using an appropriate system • mouse • Xenopus • Drosophila • zebrafish • C. elegans – not yet in chicken, most amphibians Bio. Sci 145 A lecture 11 page 14 ©copyright Bruce Blumberg 2000. All rights reserved

Embryo microinjection • • • Simple, direct way to get DNA, RNA or proteins into embryos – primary application is embryos with large cells (xenopus, zebrafish) • needles used are ~ 1 m diameter Xenopus microinjection takes two basic forms – oocyte injection – embryo injection oocyte injection – oocytes are immature eggs, do not divide – these are dissected from ovaries and can be used for various experiments – DNA must be injected into the nucleus (germinal vesicle) • transcription is possible – RNA must be injected into the cytoplasm • translation is very robust, can continue for long periods of time (days) Bio. Sci 145 A lecture 11 page 15 ©copyright Bruce Blumberg 2000. All rights reserved

Embryo microinjection (contd) • oocyte injection (contd) – applications • in vivo expression screening – microinject pools of m. RNA 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, m. RNA into cytoplasm Bio. Sci 145 A lecture 11 page 16 ©copyright Bruce Blumberg 2000. All rights reserved

Embryo microinjection (contd) • Embryo microinjection – typically performed from 1 -32 cell stage, depending on effect desired – embryos divide and develop • microinjected materials are mosaically distributed – no transcription of injected DNA before MBT • zygotic transcription begins at the midblastula stage • by then, microinjected DNA is very mosaic – transgenic approaches – RNA is well translated but less stable than in oocytes (24 -36 hrs max) – applications • misexpression of m. RNAs • injection of mutant m. RNAs • gain of function • loss-of-function – m. RNAs encoding dominant negative mutants – neutralizing antibodies – RNA-I – Morpholino antisense oligonucleotides • Can target injected materials to particular tissues by using fate maps and blastomere injections at 32 cell stage Bio. Sci 145 A lecture 11 page 17 ©copyright Bruce Blumberg 2000. All rights reserved

Embryo microinjection (contd) • Embryo microinjection (contd) – advantages • very early stages can be manipulated • targeted injections possible • possible to combine molecular biology with experimental embryology – disadvantages • no early transcription • mosaic inheritance • embryos are dividing – limited time window for injections Bio. Sci 145 A lecture 11 page 18 ©copyright Bruce Blumberg 2000. All rights reserved

Virus-mediated transfer • • • Just as with cultured cells, viral vectors may be used to express transgenes in embryos – identical viruses are used (retroviruses and adenovirus) – similar host range issues • use of retroviruses may require use of virus-free eggs (extremely expensive since most chickens carry one strain or other of RSV) clone gene of interest into viral vector – package into virions – concentrate and determine titer (infections particles/volume) – microinject into embryo applications – primary application is with chick embryo – Juan Carlos Izpisúa Belmonte advantages – relatively efficient disadvantages – no expression in early embryos! – may be impossible to express some genes • e. g. DN-RAR – retroviruses do not stay at site of injection – survival issues – non-specific effects Bio. Sci 145 A lecture 11 page 19 ©copyright Bruce Blumberg 2000. All rights reserved

Biolistic gene transfer • • Somewhat bizarre method developed for very difficult problems (plant cells) very small particles are coated with DNA – blasted into target tissue • gunpowder • compressed air advantages – works in systems that are refractory to other methods • e. g. plant cells • regenerating limbs – not very difficult disadvantages – equipment requirement – not particularly efficient • only a few % of target cells survive and take up DNA – tissues must survive partial vacuum Bio. Sci 145 A lecture 11 page 20 ©copyright Bruce Blumberg 2000. All rights reserved

Electroporation • • • Just like cultured cells, tissues and embryos can be transfected with DNA by electric pulse typical setup consists of a pair of microelectrodes (usually needles) in close proximity. – Maneuver this into close proximity of target, add DNA and zap applications – primary use is with chick embryos – some use of RF transfection in other embryos but not widely practiced or accepted advantages – can work in very early embryos – can target small areas relatively well • unlike virus-mediated transfection, the DNA only gets into cells near the electrode disadvantages – equipment requirement • electrodes must be custom made – plenty of “touch” is required – not so many applications yet • chick embryo – potential of contamination with bacteria and molds Bio. Sci 145 A lecture 11 page 21 ©copyright Bruce Blumberg 2000. All rights reserved

Transgenic technology • • Transgenesis is either not possible or not feasible in all model organisms – typical model organisms of interest are: • C. elegans • Drosophila • zebrafish • axolotl • Xenopus • chicken • mouse – transgenic techniques are well developed in • C. elegans • Drosophila • mouse – becoming reasonably doable for • Xenopus • zebrafish – not readily possible • chicken • axolotl targeted gene disruption only works in a few organisms – mouse – C. elegans Bio. Sci 145 A lecture 11 page 22 ©copyright Bruce Blumberg 2000. All rights reserved

“Standard” transgenesis - mouse • standard transgenesis – this involved microinjecting DNA into a fertilized egg (mouse) or embryo (Drosophila) • some fraction of embryos undergo integration of DNA into genome • some fraction of these transmit the transgene in the germline Palmiter et al. , 1982. Nature. 300, 611 -5. Bio. Sci 145 A lecture 11 page 23 ©copyright Bruce Blumberg 2000. All rights reserved

“Standard” transgenesis (contd) • • Each mouse that harbors a transgene and transmits it in the germline is a “founder” – founders must be evaluated before proceeding to large scale breeding and analysis • keeping mice is EXPENSIVE ~$1. 00/cage/day. – Multiple females can be caged together – but males must be kept individually • downstream analysis is very time consuming, tedious and expensive what would we like to know about a founder line? – How many copies of the transgene are present? • Prepare DNA from tails, do Southern analysis and compare with DNA standards • Transgene copy number varies from 1 to several hundred • Level of transgene expression is usually proportional to the number of copies – is the transgene expressed? Transgenes are not equally active at all integration sites. • Northern or Western analysis – Western is best but requires an antibody. » produce an antibody to the protein » engineer the transgene to express myc, flag or other common epitope – Northern is more commonly performed Bio. Sci 145 A lecture 11 page 24 ©copyright Bruce Blumberg 2000. All rights reserved

“Standard” transgenesis (contd) • 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 tissuespecific 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? – tissue Northerns – quantitative RNA blotting – RT-PCR – is the transgene transmitted faithfully? • Multiple tandem copies of the same sequences could be problematic • are expression levels similar in progeny of founders? – Same is desirable – could be more or less, or even absent Bio. Sci 145 A lecture 11 page 25 ©copyright Bruce Blumberg 2000. All rights reserved

“Standard” transgenesis (contd) • • • 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 Bio. Sci 145 A lecture 11 page 26 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting • 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 Bio. Sci 145 A lecture 11 page 27 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting (contd) How to make ES cells • ES cells are very touchy in culture – lose ability to colonize germ line with time – easily infected by “mysterious microorganisms” that inhibit ability to colonize germ line • ko labs maintain separate hoods and incubators for ES cell work – overall, ES cells depend critically on the culture conditions to keep them in an uncommitted, undifferentiated state that allows colonization of the germ line. Bio. Sci 145 A lecture 11 page 28 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting (contd) • technique – isolate genomic clones spanning the gene of interest from an ES cell library – construct a restriction map of the locus with particular emphasis on mapping the exons – create a targeting construct with large genomic regions flanking the region to be disrupted – an essential exon(s) must be disrupted such that no functional protein is produced from the gene • this should be carefully tested in cell culture before mice are made – it is often useful to design the construct such that a reporter gene is fused to the coding region of the protein • this enables gene expression to be readily monitored and often provides new information about the gene’s expression – dominant selectable marker is inserted within replacement region – negative selection marker is located outside the region targeted to be replaced – DNA is introduced by electroporation and colonies resistant to positive selection are selected. – Integration positive cells are subjected to negative selection to distinguish homologous recombinants • homologous recombinants lose this marker Bio. Sci 145 A lecture 11 page 29 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting (contd) Targeting vector electroporate recombination positive selection with dominant selective marker negative selection to identify homologous recombinants Bio. Sci 145 A lecture 11 page 30 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting (contd) Bio. Sci 145 A lecture 11 page 31 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting (contd) • • • Technique (contd) – homologous recombination is verified by Southern blotting – factors affecting targeting frequency • length of homologous regions, more is better. – 0. 5 kb is minimum length for shortest arm • isogenic DNA (ie, from the ES cells) used for targeting construct. Polymorphisms appear to matter • locus targeted. This may result from differences in chromatin structure and accessibility problems and pitfalls – incomplete knockouts, ie, protein function is not lost • but such weak alleles may be informative – alteration of expression of adjacent genes • region removed may contain regulatory elements • may remove unintended genes (e. g. on opposite strand) – interference from selection cassette • strong promoters driving these may cause phenotypes Applications – creating loss-of-function alleles – introducing subtle mutations – chromosome engineering Bio. Sci 145 A lecture 11 page 32 ©copyright Bruce Blumberg 2000. All rights reserved

Gene targeting (contd) • • • 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 Bio. Sci 145 A lecture 11 page 33 ©copyright Bruce Blumberg 2000. All rights reserved