Studying gene expression and function

Mutagenesis and protein engineering

Possible to generate mutants and determine the phenotypes of resultant proteins Can be done both in vitro and in vivo Ethylnitrourea (ENU) is used to generate mutations in vivo (mainly mice) The mutations generated are random In vitro mutagenesis enables contol of the products generated

Recombination
Three main types Homologous recombination takes place between 2 highly identical sequences Site specific recombination: between sequences that have short stretches of homology (invertible elements, resolvases, phage DNA intergration) Non-homologous (illegitimate) recombination: sequences with no homolgy at all (transiposition, xsome deletions, inversions, translocations)

Mechanism of homologous recombination

Strands align Nicks in the DNAs Strands displace to opposite DNA Ligation covalently links the DNAs (Holliday structure) The branch point migrates DNA cleaved again, new DNA molecules formed
http://www.blackwellpublishing.com/trun/artwork/Animations/Recombination/recombi nation.html

Homologous recombination and gene knock-outs

Can be used to generate a gene knock-out The method of choice for replacing one allele with an engineered construct without affecting any other locus in the genome . The DNA sequence of the gene to be replaced as well as adjoining region has to be known.

Homologous recombination and gene knock-outs

Plan to Insert different alleles (both functional and non-functional ones), Different genes or reporter genes (e.g. antibiotic resistance or green fluorescent protein). Include some flanking DNA that is identical in sequence to the targeted locus. A negative selection marker (e.g. thymidine kinase, tk) may be added to the replacement vector. The negative marker is outside the region of sequence similarity between the vector and the targeted locus.

Homologous recombination and gene

knock-outs
Then introduce construct into the cell Heat shock Electroporation Gene gun Agrobacterium Plant virus During cell division Construct aligns along the targeted gene Recombination takes place within the homologous sequences The recombination may take place anywhere within the flanking DNA sequences The exact location is determined by events in the cells.

Homologous recombination and

gene knock-outs

In this case the negative marker does not get integrated

Homologous recombination and

gene knock-outs
Cells that incorporated the construct are selected by respective antibiotic in medium (positive selection) Non-homologous recombination offspring are eliminated by addition of antibiotic as well as gancyclovir in medium Thymidine kinase activates Gancyclovir, a nucleotide analog, by addition of phosphate. Activated Gancyclovir gets incorporated, the cells die

Homologous recombination and gene knock-outs

Sucess of gene knock is affected by ploidy For haploid organisms, only one round of tranformation is necessary For diploids, 2 steps involving 2 different selection markers are desired Knock-outs in polyploids are not practical (need to use several antibiotics!!) Its difficult to target Multiple copy genes

Plan for gene knock-out from diploid cell in vitro

Make 2 constructs each with a different marker Transform with construct carrying first resistance marker Select with the respective antibiotic Transform resultant heterozygotes with second construct carrying a different marker Select with both antibiotics (use antibiotics with no known cross resistance) Confirm correct intergration by southern blots

Homologous recombination and

gene knock-outs

Gene knock-out in mice

Homologous recombination to replace one allele Followed by two or more generations of selective breeding until a breeding pair are isolated that have both alleles of the targeted gene inactivated (knocked-out). The role of a particular gene is determined by observing the knock-out phenotype.

Gene knock-out in mice

2 approaches Can transform embryonic stem cells Inject construct into newly fertilised egg

Gene knock-out in mice

Isolate developing embryo at blastocyst stage. This embryo is from a strain of mice with gray fur. Remove embryonic stem cells from grayfur blastocyst. Grow stem cells in tissue culture.

Gene knock-out in mice

Transfect stem cells with construct. Select for homologous recombination by growing stem cells in neomycin and gancyclovir.

+ neomycin

+ gancyclovir

Gene knock-out in mice

Remove homologously recombined stem cells from petri dish and inject into a new blastocyst that would have only white fur. Implant several chimeric blastocysts into pseudopregnant, white fur mouse.

Gene knock-out in mice

Mother will give birth to a range of mice. Normal white fur and Chimeric mice with some of their cells derived from recombinant stem cells. Fur cells from recombinant stem cells produce gray patches.

Gene knock-out in mice

Mate the chimeric mice with wild-type white fur mice. If the gonads of the chimeric mice were derived from recombinant stem cells, all the offspring will have gray fur. Every cell in gray mice are heterozygous for the homologous recombination.

Gene knock-out in mice

Mate heterozygous gray mice (+/ H) and genotpye the gray offspring. Identify homozygous recombinants (H / H) and breed them to produce a strain of mice with both alleles knocked out. The pure breeding mouse strain is a "knockout mouse". .

Knock-outs in plants
Discs cut from a leaf Incubated with engineered Agrobacteria for 24 hrs Selection for cells with intergrated constructs Addition of shoot inducing medium Transfer to root inducing medium Grow seedling into adult plant with transgene.

Antisence DNA oligonucleotide technology

Injecting a cell with antisense DNA oligonucleotides RNA-DNA hybrids form Degradation of hybrid by RNAse H Abolished expression of the respective protein by depleting the cytoplasm of mRNA

The antisense RNA approach

Similar effect can be achieved by engineering the cell to generate dominant negative mutations Can be done by cloning reverse complement of part of the gene in tandem ATGCTTTGCAT Resultant RNA folds onto self to make double strand This is the basis for RNA interference (RNAi)

RNA interference
A naturally occuring post-transcriptional gene silencing phenomenon Evolutionariry conserved Sequence specific We can use RNAi to answer the question: Is the gene essential?

RNA interference
RNAi is also known as knock-down Applications include In vitro and in vivo gene targeting for functional studies of genes known to be up-regulated For identification of drug targets, leading to novel therapeutic strategies

RNAi has potential in medicine

Cancers are charaterised by upregulation of groth factors and growth factor receptors Novel therapeutic interventions would target inhibition of such proteins or their down regulation

Mechanism of RNA interference (RNAi) in mammalian systems.

Mechanism of RNA interference (RNAi) in mammalian systems.

Long double-stranded RNA molecules (dsRNA), which are expressed from DNA vectors (left red arrow) or directly enter the cell (center red arrow), are processed by the Dicer complex resulting in the formation of small inhibitory RNAs (siRNAs). Alternatively, to induce RNAi these small 2123 bp duplexes are directly delivered into the cell (right red arrow). The siRNAs are incorporated into a nucleasecontaining multiprotein complex called RISC, which becomes activated upon the ATP-dependent unwinding of the siRNA duplex by an RNA helicase. The now single-stranded siRNA guides the RISC complex to its complementary target mRNA which is then cleaved by the endonucleolytical activity of RISC. While the RISC complex is recovered for further cycles, the cleaved mRNA molecule is rapidly degraded due to its unprotected RNA ends.

Dicer and RISC (RNA-induced silencing complex). RNAi is initiated by the Dicer enzyme (two Dicer molecules with five domains each are shown), which processes double-stranded RNA into 22-nucleotide small interfering RNAs36. Based upon the known mechanisms for the RNase III family of enzymes, Dicer is thought to work as a dimeric enzyme. Cleavage into precisely sized fragments is determined by the fact that one of the active sites in each Dicer protein is defective (indicated by an asterisk), shifting the periodicity of cleavage from 9 11 nucleotides for bacterial RNase III to 22 nucleotides for Dicer family members40. The siRNAs are incorporated into a multicomponent nuclease, RISC (green). Recent reports suggest that RISC must be activated from a latent form, containing a double-stranded siRNA to an active form, RISC*, by unwinding of siRNAs41. RISC* then uses the unwound siRNA as a guide to substrate selection31. b, Diagrammatic representation of Dicer binding and cleaving dsRNA (for clarity, not all the Dicer domains are shown, and the two separate Dicer molecules are coloured differently). Deviations from the consensus RNase III active site in the second RNase III domain inactivate the central catalytic sites, resulting in cleavage at 22-nucleotide intervals.

Small interfering RNAs versus small temporal RNAs Double-stranded siRNAs of length 2123 nucleotides are produced by Dicer from dsRNA silencing triggers. Characteristic of RNase III products, these have two-nucleotide 3' overhangs and 5'-phosphorylated termini. To trigger target degradation with maximum efficiency, siRNAs must have perfect complementarity to their mRNA target (with the exception of the two terminal nucleotides, which contribute only marginally to recognition). stRNAs, such as lin-4 and let-7, are transcribed from the genome as hairpin precursors. These are also processed by Dicer, but in this case, only one strand accumulates. Notably, neither lin-4 nor let-7 show perfect complementarity to their targets. In addition, stRNAs regulate targets at the level of translation rather than RNA degradation. It remains unclear whether the difference in regulatory mode results from a difference in substrate recognition or from incorporation of siRNAs and stRNAs into distinct regulatory complexes.

The degradation of endogenous mRNA in a sequence-specific manner can be induced by dsRNA [RNA interfernce (RNAi)], antisense transcription, or viral infection. In the current model for posttranscriptional gene silencing by RNAi, the ribonuclease III like enzyme Dicer processes dsRNA into small fragments (siRNA) of 21-25 nts. The siRNA is incorporated into the RNA-induced silencing complex (RISC), which targets and degrades the homologous mRNA. Most of the proteins in the RISC (with the exception of Ago2 and Dicer have not been identified) but likely contain endonuclease, helicase, exonuclease and homology scanning activity.

Delivery systems for RNAi

Constructs incorporated into Liposomes: Lipid micelles that fuse with cell membranes contents get delivered to the cytosol Polyethylenimine

Then injected in mammal Intraperitoneally, Intravenous Intrathecal Intratumoral, e. t. c

Polyethylenimine (PEI)-mediated siRNA transfer

Polyethylenimine (PEI)-mediated siRNA transfer

Upper panel: PEIs are synthetic linear (a) or branched (b) polymers with an amino group in every third position. Dependent on the pH, some of these amino nitrogens are protonated giving PEI a high cationic charge density. Lower panel: proposed mechanism of PEI-mediated siRNA transfer. Due to electrostatic interactions, PEI is able to complex negatively charged siRNAs leading to a compaction and the formation of small colloidal particles which are endocytosed. The proton sponge effect exhibited by PEI complexes leads to osmotic swelling and ultimately to the disruption of the endosomes. siRNAs are protected from degradation due to their tight condensation in the complex and the buffering capacity of PEI. Upon their release from the PEI-based complex, intact siRNAs are incorporated into the RISC complex and induce RNAi

T. brucei as a model for RNAi

MARKER

MARKER

AAAA

Inducible stem-loop or opposing T7 promoters DsRNA processed by ds endonuclease to 21nt fragments with short overhangs) Ds fragments in RISC complex; target RNA by base-pairing Target RNA destroyed

The first RNAi in T. brucei 1998

Double-stranded RNA with alpha tubulin sequences in trypanosomes Cells get round and stop growing

Advantages of RNAi
Quick Simple Can target multi-copy genes even if they are spread around the genome

Disadvantages of RNAi
The RNA and protein are not eliminated
Very often 10-15% of the normal level is sufficient. If that is how the RNAi works, no change in phenotype will be seen.

Leakiness - control by tetracycline may be incomplete

Often there is some dsRNA made even in the absence of tetracycline. If a small reduction in the protein has deleterious effects, no live cells will be obtained at all.

Disadvantages of RNAi
Escapers from deleterious RNAi
After tetracycline is added, the cells initially show effects but after about 5 days, they recover and look normal During the initial selection, cell lines are obtained but the RNAi mysteriously doesnt work. The cell lines look great for a couple of weeks of culture but after that the RNAi doesnt seem to work any more The cell lines look great and are frozen down. When you thaw them out the RNAi no longer works.