Techniques

3 ways of generating siRNAs in vitro for mammalian cells

1. Artificially synthesise complementary 21-mer RNA oligos and anneal them together 2. Transcribe both strands of a short DNA sequence in vitro using phage RNA polymerase (eg. T7) then anneal the complementary RNAs 3. Produce long dsRNA sequence in vitro (using one of the mechanisms described above but generating longer DNA sequence) then treat with Dicer before use (produces a gene specific cocktail of siRNAs)

Advantage of RNA-seq

1. Can accurately measure transcription of poorly expressed and very highly expressed genes (wide dynamic range) - microarrays and chips have a much narrower range 2. Can distinguish between alternatively spliced mRNA variants from the same gene 3. Can distinguish between transcripts from different alleles of the same gene (very similar sequences, difficult to distinguish using microarray)

The biological role of co-suppression

1. Helps protect the cell against RNA viruses that replicate via a dsRNA intermediate 2. Helps protect against transposable element movements that involve dsRNA intermediates 3. miRNA used in development

RNA-Seq

1. Isolate mRNA using oligo dT (short ssDNA made of dTTP, which polyA tail of mRNA will hybridise to, whereas rRNA and tRNA wont) 2. Fragment mRNA and convert to cDNA using random primers (RT-PCR) 3. Sequence cDNA (NGS) 4. Map each sequence to a region of the genome (bioinformatics) 5. Measure number of reads per gene (number of cDNA molecules per genes, so number of mRNA fragments per gene, which is proportional to the amount of mRNA per gene, telling you how highly expressed the gene is)

Types of gene expression assay

1. Measure protein product - Reporter assay, Western blot 2. Measure steady state level of mRNA - Northern blot, RT-PCR, qPCR, microarrays/gene chips, sequencing assays (RNA-Seq) 3. Measure mRNA at certain time point - Nuclear run-on assay

Techniques used in functional genomics

1. PCR based gene targeting (yeast, DNA) 2. Anti-sense morpholinos (animal embryos, mRNA) 3. RNAi (C. elegans, mammalian cell culture) 4. The CRISPR-Cas9 system (animals, plants and fungi)

2 ways anti-sense morpholinos can inhibit gene expression

1. bind to mRNA and block transcription by blocking progress of transcription complex 2. bind to pre-mRNA splice sites (intron/exon boundary), modifying splicing and mRNA production

qPCR

A form of RT-PCR. Measures how the amount of product changes with each PCR cycle by measuring fluorescence by reporter probe oligonucleotide e.g. TaqMan, or SYBR green which binds to dsDNA. Amount of fluorescence emitted is equal to the amount of reporter probe bound to the target DNA, and so measures the amount of DNA at each cycle (increase in fluorescence over time). Plot a standard curve of threshold cycle (CT) against the log of the number of mRNA molecules initially in each sample, and use this standard curve to calculate amount of mRNA from CT value of unkown sample

RT-PCR

A technique in which RNA is first converted to cDNA by reverse transcriptase (after addition of primer 1), then the cDNA is then converted into dsDNA in the first cycle PCR (using primer 2). In subsequent cycles the dsDNA is amplified (using primer 1 and 2). PCR reactions only linear for initial cycles, however 20-30 cycles needed for visible band, so not quantitative

RNA interference (RNAi)

A technique used in functional genomics to silence the expression of selected genes. RNAi uses synthetic dsRNA molecules (200nt+) corresponding to the gene of interest. This leads to the dramatic reduction in protein product from this targeted gene

Protein purification by affinity tag (e.g. polyhistidine-tag)

Affinity tag incorporated into target protein. Can be added onto N terminus or C terminus of the protein (depending on which end it wont disrupt the protein function). Tag binds strongly to Ni2+ ions. Protein moves down column, when comes into contact with the nitriloacetic acid (NTA) it binds because of the histidine residues. Other proteins don't have His tag so pass through the column. Then elute the His tag protein from the column using imidazole

ChIP-Seq

An alternative to ChIP-chip. DNA fragments isolated by ChiP are identified by high throughput sequencing, rather than from hybridisation to a microarray

Secondary antibody

An antibody which specifically binds to a primary antibody, and is linked to an enzyme such as alkaline phosphatase, or horseradish peroxidase, which catalyse the production of a coloured precipitate from a particular substrate. Amount of colour is a measure of the amount of secondary antibody, and the amount of protein, present

PCR-based gene disruption in yeast

Based on homologous recombination, so works well in yeast. A plasmid eg. pFA6A-KanMX4 (containing selectable marker e.g. Amp R) is used as a template for PCR. PCR primers are added that are complementary to DNA flanking the kanMX4 gene. Extra sequences have been added to the 5' end of the primers, which are homologous to sequences flanking the target yeast gene. Therefore, the PCR product amplified will be the kanMX4 gene with sequences incorporated at both ends that allow homologous recombination. Introduce PCR product into yeast cells (identified using ampicillin), and homologous recombination can occur. In the event of a double cross-over (recombination events on either side of the kanMX4 gene), the target gene is knocked out and replaced with the kanMX4 gene. KanMX4 is a bacterial gene but it links up with a promoter in yeast, so is expressed in yeast cells

How is RNAi triggered in C. elegans and mammalian cells?

C. elegans - Worms can be injected with long dsRNA, fed bacteria expressing a specific dsRNA (T7 promoter at both ends of gene so that both strands transcribed) or soaked in a solution containing dsRNA Mammals - Long dsRNAs cannot be used because they produce an anti-viral response (general reduction in translation for all types of RNA and general RNA breakdown). To avoid this, individual siRNAs (generated in vitro) are introduced or expressed directly (expressed in vivo, more common approach)

DNA-calcium phosphate co-precipitation

Cells are washed in phosphate buffer, DNA is added and precipitated using calcium chloride. DNA precipitates are internalised by endocytosis

Minimal promoter

Core promoter upstream of reporter gene. MCS upstream of core promoter, into which DNA fragment can be inserted, which may contain binding site for specific transcription factors (enhancer/proximal promoter)

ChIP-on-chip - Identification of genome wide identification of protein:chromatin interactions

Custom made microarrays can be used to identify all the genome binding sites for a known protein. The microarray contains sequences corresponding to intergenic regions (where regulatory proteins are likely to be bound. Microarrays are normally intragenic). DNA fragments isolated by ChIP are labelled and hybridised to microarray. The sequences they bind to correspond to genome regions bound by the transcription factor/chromatin in vivo (at the time the formaldehyde was added)

Anti-sense morpholinos

DNA bases, complementary to the mRNA being silenced, attached to a backbone of morpholine rings (instead of sugar) linked through phosphorodiamidate groups. Single-stranded. Typically made as exact match 25 mers overlapping the first AUG (translation start codon) in the target mRNA

hormone response element (HRE)

DNA sequence within the promoter of a gene, with a specific attachment site for a given steroid hormone and its nuclear receptor (hormone receptor complex)

Methods of gene transfer into animal cells

Direct DNA transfer (not used that often) Micro-injection Gene gun (most often used in plant genetic manipulation) Chemical DNA-calcium phosphate co-precipitation Liposome-mediated Physical Electroporation Viral (Higher transfection efficiency than chemical/physical, potentially 100%) Retroviral Adeno-associated viral

Contrast the different methods of site-directed mutagenesis

Early methods ssDNA template plasmid with 1 primer containing mutation dut ung method - template DNA containing U digested by dUTPase In vivo amplification by E. coli QuikChange method dsDNA template plasmid with 2 primers containing mutation template DNA contain methyl A digested by Dpn1 Mutant plasmid made in vitro and then amplified in vivo Modern method ordering gBlocks Gene Fragments is a lot less expensive that trying to create mutant DNA on your own

Describe how ChiP works

Express epitope tagged protein in target cells e.g. yeast cells. This tagged regulatory protein binds to specific site in the genome e.g. promoter. Treat cells with formaldehyde to stabilise protein (crosslinks proteins to proteins, and proteins to DNA). Isolate DNA + linked proteins using antibody that recognises. Break up DNA into smaller pieces (so that some of these pieces have epitope tagged protein bound). Reverse protein-DNA cross-links (by heating up the mixture) then identify the presence of individual DNA fragments using PCR (using specific primers for DNA fragments you are looking for)

How to create cell lines

First dissociate cells from a tissue and then put in growth medium. This creates a primary culture. This culture is subcultured into separate flasks to create primary cell lines. These either eventually senesce, or are immortalised/transformed to establish a permeant cell line

Commonly used mammalian cell lines

HeLa, NIH3T3, P19, COS-7

Imidazole

Imidazole similar in shape to histidine so competes with histidine for the binding to the Ni NTA, dislodging the His tag protein so it can then move down the column and be eluted

Immortalised vs Transformed mammalian cell lines

Immortalised - cells have mutations to allow continuous growth in culture but do not have the properties of cancer cells Transformed - immortalised cells that have the properties of cancer cells

Western blotting

Immunoblotting. Uses primary and secondary antibodies to detect the presence of a particular protein. Proteins separated using SDS-PAGE, then transferred onto nylon membrane. A primary antibody (from mice) binds to band containing protein of interest. A secondary antibody (from rabbits), labelled with an enzyme, detects the bound primary antibody. Amount of colour changed caused by enzyme is a measure of the amount of secondary ab present, and this the amount of protein present in the sample

RNA Polymerase III

In eukaryotic cells, transcribes DNA to synthesise a variety of small RNAs: 5S rRNA, tRNA, SINEs(?) and other small RNAs including siRNA. Pol III terminates once reaches small stretch of polyT

How are SiRNAs generated in vivo for mammalian cells?

Introduce DNA encoding the required SiRNA into mammalian cells using plasmid vector. RNA expression driven by a RNA polymerase III promoter (sequence you want to be transcribed is placed down stream of a Pol III promoter. Pol III terminates once reaches small stretch of polyT, so can make PolIII stop transcribing at exact point) The DNA encodes a single RNA molecule with complementary sequences. The encoded RNA folds into a ds form to form hairpin loop, which is then processed

Techniques used to analyse protein function

Making DNA mutations 1. Random PCR mutagenesis 2. Site directed mutagenesis (early methods e.g. dut ung method, QuikChange method, modern method, alanine scanning) Measuring effect of mutant protein 1. physiological expression in vivo 2. purify mutant protein to test the effects of a large quantity of the protein in vitro

Normalisation

Measure the amount of mRNA corresponding to a gene known to be expressed at the same level in all the samples, typically a housekeeping gene (eg Beta Actin) - internal standard. If the apparent amount of mRNA corresponding to this housekeeping gene changes in the samples then the results for the experimental gene can then be corrected (normalised) to allow for this. Important when comparing a range of different samples in qPCR to prevent differences in the amount of starting cells, efficiency of extraction etc. skewing results.

Dicer

Member of RNAse III family and part of RISC. Enzyme that cleaves and processes dsRNA to produce siRNAs or miRNAs. Requires ATP

Why is the effect of anti-sense morpholinos long lasting?

Morpholinos are more stable than DNA. If introduced into a living cell it is not degraded by the same enzymes as for DNA/RNA (DNAses/RNAses), so the RNA silencing is long lasting, so can see the effects over a period of time of blocking protein synthesis

Why are higher-eukaryotic cells (e.g. mammalian cells) difficult to grow in culture?

Most cell types only undergo a limited number of divisions before dying

Pros and cons of the different ways to measure steady state level of mRNA

Northern blotting Widely used as straight forward and quantitative Low sensitivity - can't detect RNA from lowly expressed gene/small sample of RNA. Need sufficient RNA for band to show RT-PCR PCR reactions only linear for initial cycles, however 20-30 cycles needed for visible band, so not quantitative qPCR More sensitive than RT-PCR - allows quantitative analysis as allows the measuring amount of PCR product after just a few cycles. TaqMan quencher doesn't always totally inhibit fluorescence from fluorophore which can give false results. SYBR green binds only to dsDNA, and is non-specific. Microarrays/gene chips/sequencing assay Measure the expression of many genes at once. Gene chips more expensive, but more expensive and consistent than microarrays. More recently RNA-Seq has replaced microarrays/gene chips as it has high resolution (distinguish between alleles and alternatively spliced variants) and wide dynamic range

Disadvantage of anti-sense morpholinos

Not perfect technique, doesn't work with total specificity. Sometimes the morpholino can bind to other types of mRNA and inhibit the translation of several different proteins. So ideally, morpholino experiment done first, then results are confirmed using conditional gene knockout experiment

Co-suppression / quelling

Phenomenon seen in plants and certain fungi. Introduction of extra copies of a gene (as transgenes) leads to "turning off" of both the transgenes and the existing copies of the same gene on the chromosomes. Results from the production of dsRNA as a consequence of aberrant transcription

Polyacrylamide gel vs agarose gel

Polyacrylamide gels have smaller pores, so allow smaller DNA fragments (1−1000bps), and proteins, to be separated. Agarose gels can be used to resolve large fragments of DNA

Nuclear receptor

Protein inside a eukaryotic cell that, on binding to a specific hormone, forms a hormone receptor complex. This then enters the nucleus and regulates transcription by binding to hormone response elements

Northern, Southern, and Western blotting

RNA, DNA, proteins

Pros and cons of the different ways to measure protein expression

Reporter assay Can be used for living cells e.g. GFP Western blot More quantitative

Alanine scanning

Systematically change amino acids within a functional domain to alanine, and measure the effect of each mutation on protein function. Alanine has simple R group (CH3), so effect seen is due to loss of more complex R group rather than gain of anything else, which gives information about the function of the original amino acid

Random PCR mutagenesis

Taq polymerase - approx 3-6 errors per 100kb. Increasing the concentration of Mg2+ and adding Mn2+ ions increases the error rate of Taq polymerase. Also, changing the ratio of dNTPs so that unequal also increases error rate. Mutant PCR produced produced are sequenced to work out which ones will be useful and what effect those mutations have

TaqMan qPCR

TaqMan probes are about 20 nucleotides long and have a fluorophore at the 5' end, and a quencher at the 3' end. Energy released by fluorophore is absorbed by the quencher, so probe does not fluoresce. The reporter probe is complementary to the top strand. Primer is added which is complementary to the 3' end of the top strand (also bottom strand). Taq polymerase extends this primer from its 3' end until reaches reporter probe, which is when it uses its 5'-3' exonuclease activity to degrade reporter probe and release fluorophore. The fluorophore emits energy on excitation, which is not absorbed by the quencher. Amount of fluorescence emitted is equal to the amount of reporter probe bound to the target DNA, and so measures the amount DNA at each cycle (increase in fluorescence over time)

How does RISC cleave target mRNA?

The PIWI domain of the Ago protein has acts as a ribonuclease ("Slicer") which cleaves the target RNA and destroys it. Cleavage occurs approximately in the middle of the region where the guide RNA is paired to the target RNA

Green Fluorescent Protein (GFP)

The jellyfish A. victoria contains both GFP and the protein aequorin, which emits light. This light is absorbed by GFP which then fluoresces green. Expose to UV light causes GFP to fluoresce in the absence of aequorin. If GFP is attached to a protein of interest, then the level of fluorescence in UV light indicates the amount (and location) of your protein in a cell

CT value

The threshold cycle for qPCR. The cycle at which the amount of fluorescence exceeds a set threshold. This threshold is set low so that PCR is still in the linear stage

Transient vs stable transfection

Transient DNA taken up by cells will be maintained for a short period of time (24 - 48 hrs) before it is degraded and/or diluted from the cell during division. However the time is sufficient to express genes from the vector DNA providing a rapid way to analyse gene expression and protein function Stable The recombinant vector is maintained as an episomal replicon in the host cell. If this integrates into the host genome, a stable cell line is created

Why must animal cells be treated to increase the efficiency of transfection?

Uptake of DNA by animal cells is a very rare event

How to construct a microarray

Use PCR to amplify specific cDNA corresponding to the genes you want to analyse (by using specific primers, dsDNA synthesised). Each PCR product (dsDNA) is spotted in a specific position on surface of glass slide (coated in poly-lysine, allowing DNA to be fixed in position). UV treatment crosslinks the DNA with the lysine, fixing it onto the glass slide. The dsDNA is denatured make single-stranded. ssDNA can hybridise with complementary labelled cDNA made from the mRNA you want to detect

Chromatin immunoprecipitation (ChIP)

Used in combination with microarrays and sequencing assays. Widely used for the analysis of interactions between regulatory proteins and the genome - e.g. identification of transcriptional activator protein binding at promoters and enhancers. Often uses an epitope tagged protein to allow immunoprecipitation, isolating the protein and the DNA it's bound to. DNA fragments isolated by ChiP can be identified by RNA-Seq or hybridisation to a microarray/gene chip.

Early method of site-directed mutagenesis (70s and 80s)

Uses single stranded circular template DNA (like ssDNA of M13 bacteriophage) and a short complementary primer containing the required mutation. Primer extended using DNA polymerase and dNTPs. Double stranded molecules produced have one mutant strand with a "nick". Introduce these molecules into E. coli and the nick is repaired. The DNA is replicated by the E. coli - approx half of the resulting molecules will have two mutant strands (other half 2 normal strands)

QuikChange method

Whole plasmid PCR method of site-directed mutagenesis using a commercial kit. Uses ds plasmid DNA with methylated A as a template and two complementary PCR primers both containing the required mutations. Pfu polymerase used so that unwanted mutations are not introduced (12-18 = few cycles to prevent mutant too). After one PCR cycle, the molecules have 1 mutant strand (heteroduplex) and 1 nick. After 2 cycles, molecules with 2 mutant strands (both with nicks) are generated (All primers mutant so new unmutated strands can't be created). Heteroduplex molecules created after 1st cycle are cut using Dpn1 which recognizes the sequence GATC only where A is methylated. Therefore after Dpn1 treatment only plasmids with 2 mutant strands. These are introduced into E. coli which repairs the nicks and replicates the molecules

Microarrays vs gene chips

Work in an almost identical way (create cDNA from mRNA sample which hybridises to surface), however synthesised in slightly different ways Microarrays - use cDNA on a glass slide Gene chips - use oligonucleotides on a silica surface of a chip (the oligos are biased towards the 3' end of the gene, so detecting cDNA made from full length transcripts) Also Microarrays less sensitive and consistent. However they are relatively cheap and straightforward to use. They can be custom made by individuals in a lab, whereas gene chips are only available as a commercial product

Bacterial expression vector

a cloning vector that contains a highly active regulatable bacterial promoter just upstream of a MCS/polylinker where a eukaryotic gene (no introns as those can't be processed by bacteria) can be inserted. Contains selectable marker so can select for bacteria that have taken up the plasmid DNA

SDS

a detergent that denatures proteins, and coats them making them negatively charged so can be separated by electrophoresis

Pfu polymerase

a high-fidelity heat stable DNA polymerase. About 10x more accurate than Taq polymerase as it has 3'-5' exonuclease proof-reading activity

COS-7 cells

african green monkey kidney cells, SV40 transformed (circular ds DNA virus, produces large T-antigen which inhibits p53 - a key negative regulator of cell division)

Dut ung method

an early method of site-directed mutagenesis. Earlier methods were inefficient due to the repair of the heteroduplex region by E. coli, causing the loss of the mutation. The dut ung method if far more efficient, and leads to the non-mutant strand being destroyed in E. coli so all the molecules produced by DNA replication have two mutant strands. (Only the template DNA strand contains U, and U-containing strands targeted for degradation in ung+ E. coli, digested by dUTPase).

pET plasmid

an expression vector containing T7 promoter, lac operon, and lacI gene. Lac operon inserted near start of transcription at T7 promoter, such that the lac repressor blocks the transcription at start site in promoter by binding to operator. Therefore target gene switched off in absence of lactose or IPTG.

IPTG

analogue of lactose

SV40

circular ds DNA virus, produces large T-antigen which inhibits p53 - a key negative regulator of cell division, therefore can cause tumours in hosts

siRNA

class of dsRNAs about 21-25 nucleotides in length that silence gene expression. They contain two unpaired nt at the 3' end of each strand. The top strand is the passenger strand (which is degraded), and the bottom strand is the guide strand which associates with RISC

gBlocks Gene Fragments

commercially available synthetic dsDNA fragments with a specific DNA sequence, 125 - 3000 bp. 3kb is sufficient to encode most eukaryotic proteins without introns (median protein size in humans is 375 amino acids ~ 1kb)

What is the cDNA (from the mRNA you want to detect) on a microarray labelled with?

commonly labelled with a fluorescent dye such as Cy3 or Cy5. Cy3 is excited at 550nm and emits green/yellow fluorescence. Cy5 is exited at 650nm and emits red fluorescence. The dye is made to contain a chemical group (NHS-ester group) which reacts with an aminoallyl group incorporated into a modified nucleotide added to the cDNA synthesis reaction Can use both dyes at the same time in CGH

RISC

contains Dicer enzyme that cleaves dsDNA to produce siRNA. containing Ago protein - Paz domain binds the two nucleotide 3' overhang of the siRNA, anchoring it to the RISC complex. The siRNA duplex is unwound in an ATP-dependent. After passenger strand degraded and guide strand binds to target mRNA, PIWI (endonuclease) domain of Ago protein cleaves the target RNA

What does transformation mean in the context of mammalian genetics?

conversion of a normal cell to a cancer cell, not the same as transformation in bacterial genetics. That kind of transformation is known as transfection in mammalian genetics to avoid confusion

formaldehyde

crosslinks proteins to proteins, and proteins to DNA, therefore it fixes the DNA binding protein to the region of DNA it was interacting with when experiment performed

siRNA pathway of post-transcriptional gene silencing

dsRNA made in the nucleus is exported to the cytoplasm. It is then cleaved by an enzyme called Dicer to generate siRNAs. The siRNA duplex is incorporated into a RNA-induced silencing complex (RISC). The siRNA duplex is unwound in an ATP-dependent step, one strand remains associated with RISC (guide strand) the other is degraded (passenger strand). The single strand RNA in the RISC complex then targets it to a complementary mRNA (by base pairing) which is destroyed by the PIWI domain of the Ago protein in RISC

pLysS

encodes T7 lysozyme protein, which degrades T7 RNA polymerase. Some T7 RNA polymerase is still transcribed in the absence of IPTG, so this ensures that T7 gene 1 not expressed in absence of IPTG. In the presence of IPTG, the large amount of T7 RNA polymerase transcribed completely oversaturates the lysozyme, so there is plenty of T7 RNA polymerase to bind to the T7 promoter in pET plasmid and transcribe target gene

lacI

encodes the lac repressor. Binds to operator in the lac operon, keeping lac operon switched off in absence of lactose

T7 promoter

from T7 bacteriophage, used extensively in molecular biology due to its extremely high affinity for T7 RNA polymerase and thus high level of expression

Northern blotting

gel electrophoresis of mRNA followed by hybridization with a probe on a membrane. Transfer RNA from an agarose gel to a nylon membrane, and incubate with a radioactively labelled ssDNA probe. Black band formed on X-ray film corresponding to radioactive DNA, intensity of band is a measure of the amount of RNA

Regulated promoter

hormone response elements, upstream of core promoter, allow the core promoter to be switched on only in the presence of the hormone. Downstream of core promoter is MCS, into which target gene inserted.

HeLa cells

human epithelial cancer cells of a strain maintained in tissue culture since 1951 and used in research, especially in virology. Derived from Henrietta Lacks, who died of cervical cancer in 1951, taken without her consent

NIH3T3 cells

immortalized mouse fibroblasts (connective tissue cells)

How is the function of a gene in heterozygous knockout diploid yeast cells identified from PCR-based gene disruption?

kanMX4 confers resistance to kanamycin and G418 (which is an antibiotic very similar to kanamycin). Target gene in yeast replaced with kanMXF gene. KanMX4 is a bacterial gene but it links up with a promoter in yeast, so is expressed in yeast cells. Sporulate diploid so that each cell produces 4 haploid spores (inside ascus). If the deletion is not disrupting an essential gene, there will be 4 spores (2 G418 res, 2 G418 sens). However, if the disrupted gene is essential, then only 2 out of the 4 spores will be able to germinate (2 G418 sens)

unc22 gene

low expression of this gene causes the twitching phenotype in C. elegans. This phenotype can be triggered by RNAi

Reporter assay

measure the expression of a gene by measuring the amount of protein product produced under certain promoter region. Reporter genes include GFP, lacZ, luciferase

Give 4 basic promoters in mammalian expression vectors (RNA Pol II promoters)

minimal, constitutive, cell-specific, regulated

P19 cells

mouse embryonal carcinoma cell line

Give 3 selectable marker genes used in the transfection of mammalian cells

neo (geneticin/G418), hyg (hydromycin B), pac (puromycin). All allow cells to grow in the presence of chemicals that normally block protein synthesis/cell growth.

Modern method of site-directed mutagenesis

order mutant DNA sequence e.g. gBlocks Gene Fragment. A lot more cost effective than doing it on your own

how can the pET plasmid be used to tightly regulated protein expression in E.coli

pET introduced into E. coli strain. The E. coli contains lacI, and the TG gene 1 that codes for T7 RNA polymerase. TG gene 1 is under the control of the lac promoter. The lac repressor from the lacI gene binds to the lac operator downstream of lac promoter, keeping T7 gene 1 switched off in absence of lactose or IPTG. pLysS degrades T7 RNA polymerase in the absence of IPTG. Upon addition of IPTG, the Lac repressor then dissociates from lac operator in E.coli genome, allowing E. coli RNA polymerase to transcribe the T7 gene 1 which encodes T7 RNA polymerase. The lac repressor also dissociates from the lac operator in the pET plasmids, allowing T7 RNA polymerase to transcribe the target gene.

Episome vs plasmid

plasmids are extra-chromosomal DNA that cannot link up with chromosomal DNA, whereas episomes can e.g. viral episomes

Constitutive promoter

proximal promoter, upstream of a core promoter, contains specific transcription factor binding sites found in all cell types. Downstream of core promoter is MCS, into which target gene can be inseted. Constitutive promoter used to direct unregulated expression of gene products, as proximal promoter is turned on all the time, irrespective of environment and developmental factors

Cell-specific promoter

proximal promoter, upstream of a core promoter, contains specific transcription factor binding sites only found in some cell types. Downstream of core promoter is MCS, into which target gene inserted. Gene product expressed only in certain cell types

Chalcone synthase

purple colour gene in Petunia. First gene in which the phenomenon of RNA interference was observed; researchers, intending to upregulate the production of pigments, introduced a transgene for chalcone synthase. Instead the transgenic plants had mottled white flowers, indicating that the introduction of the transgene had downregulated or silenced chalcone synthase expression

How could lacZ be used as a reporter gene?

replace coding region of gene with lacZ which encodes β-galactosidase. 3'UTR from gene remains, which microRNA can bind to. Can easily measure the activity of β-galactosidase, which is proportional to amount of original gene product

Dpn1

restriction enzyme that recognizes the sequence GATC only where A is methylated. Used in the QuikChange method to degrade heteroduplex molecules formed in the PCR cycles

RBS

ribosome binding site. Added to expression vector, so that it is incorporated into the mRNA transcribed, ensuring that mRNA is recognised by ribosome and is translated into protein

Heteroduplex region

segment of DNA containing a mismatch, where the complementary DNA strands are very slightly different. E. coli will repair this heteroduplex region

dsRNA formation

siRNA - caused by transcription of both strands of the same region of DNA. These complementary mRNA molecules can then hybridise miRNA - dsRNA can also form when an inverted repeat is transcribed and intramolecular base paring occurs, forming a stem-loop structure

Site-directed mutagenesis

the ability create a mutation in one or a few specific bases in a DNA fragment, rather than random bases

Why is the early method of site-directed mutagenesis, before the dut ung method, inefficient?

the molecules introduced into E. coli have a heteroduplex region which is preferentially repaired on the mutant strand, so the mutation is lost before replication

What happens after an embryo is injected with anti-sense morpholinos?

the morpholinos bind to complementary mRNA, usually at the transcriptional start codon, blocking the production of a particular protein in one cell or a small number of cells. The effect on the future development of the embryo is then measured. Useful tool for studying the importance of proteins in developmental processes in specific tissues in developing embryos

Epitope

the part of an antigen molecule to which an antibody (commerically available) attaches itself. Used in ChiP for immunoprecipitation and isolation of the protein and the DNA it's bound to. The epitope tag is usually put at one terminus of the protein to avoid disrupting function Myc tag = GluGlnLysLeuIleSerGluGluAspLeu (comes from myc oncoprotein) Flag tag = AspTyrLysAspAspAspAspLys (artificial)

Functional genomics

the study of the relationship between genes and their function. Used to identify the function of new genes identified by genome sequencing projects. Usually done by inhibiting gene function and looking at the resulting phenotype