Gene Knockdown and Overexpression Systems PDF

Non – Coding RNA molecules in cell signalling / Gene knockout/down and overexpression systems Thomas Illingworth Week 1 [email protected]  RNA – a multi functional molecule (1)  RNA...

Non – Coding RNA molecules in cell signalling / Gene knockout/down and overexpression systems Thomas Illingworth Week 1 [email protected]  RNA – a multi functional molecule (1)  RNA forms an important part of the central dogma of molecular biology  Over 75% of the human genome is transcribed to RNA  However, less than 2% of that transcribed RNA produces protein coding transcripts  The remaining RNA are non – coding transcripts Sensitivity: Internal  RNA – a multi functional molecule (2) Table found in Cech and Steitz 2014 doi: http://dx.doi.org/10.1016/j.cell.2014.03.008 Sensitivity: Internal  Non – coding RNA (ncRNA) classification (1)  Ribosomal RNA (rRNA)  rRNA is the major structural component of the ribosome and has sequence complementarity to regions of mRNA with which it can interact. There are 4 rRNA molecules used as standards in eukaryotes.  Transfer RNA (tRNA)  Involved in translation of mRNA signals into proteins  Small nuclear RNA (snRNA)  Two classes, smRNA and lsmRNA named due to the type of protein they bind, and have roles in pre-mRNA modification. Sensitivity: Internal  Non – coding RNA (ncRNA) classification (2)  Small Nucleolar RNA (snoRNA)  Guides chemical modification of other RNA based molecules like rRNA, tRNA nad smRNA.  Piwi interacting RNA (piRNA)  Largest class of small non coding RNA. Primarily involved in gene silencing, particularly within repeating transcripts in mammalian germlines  Short interfering RNA (siRNA)  Gene silencing: cleavage of RNAs derived from viruses, retroelements and repeat sequences Sensitivity: Internal  Non – coding RNA (ncRNA) classification (3)  Micro RNA (microRNA)  Gene silencing: translational repression or cleavage of target mRNAs  Extracellular RNA (exRNA)  Found within bodily fluids in eukaryotes, is believed to be involved in cell- cell communication and cell regulation  Small Cajal body-specific RNA (scaRNA)  Localised to a nuclear organelle the Cajal, and regulates the activity of RNA polymerase II transcribed spliceosome RNA’s  This list is not exhaustive, there are multiple different classifications of ncRNA’s Sensitivity: Internal  Long non-coding RNA (LncRNA) (1)  LncRNA are the most common group of ncRNA’s and are defined as being over 200 bases in length  They regulate gene expression and protein function  Cis or trans acting transcriptional regulatory elements contain recognition sites for trans acting DNA binding transcription factors, which function to either enhance or repress transcription.  For example Xist, deactivates X chromosome function by tethering to polycomb-repressive complexes found on the X chromosome in cis confirmation Sensitivity: Internal  Long non-coding RNA (LncRNA) (2)  Loss of regulation of LncRNA’s has been observed in human cancers, with overexpression of a number of LncRNA’s (eg:HOTAIR) being associated with metastasis  HOTAIR can induce metastasis via acting as a tether linking EZH2/PRC2 to LSD1.  In addition, LncRNA’s have also been described as tumour suppressor genes and oncogenes.  Both of these characteristics suggest that they may be useful as cancer biomarkers in the future.  Either for diagnosis or maybe even for treatment of cancer. Sensitivity: Internal  How does this link to lab techniques I hear you cry!  Non-coding RNA’s represent the large proportion of transcribed RNA within eukaryotic cells  RNA molecules can directly and indirectly effect gene expression, working within signal pathways to promote the desired effect  Non-coding RNA molecules, as well as other different techniques can be manipulated to the researchers needs! Sensitivity: Internal  Laboratory approaches to cell signalling study  Changing the expression profile of a particular cell can give insight onto how cell signalling mechanisms are regulated.  Overexpression and gene knockouts/knockdowns have become a big part of the toolkit used to study cell signalling pathways  In this session we will go through the various methodologies which can be used to produce knockdown and overexpression systems for study. Sensitivity: Internal  Gene knockout  As the name would suggest, the process involves the permeant deactivation of a specific gene within an organism  The process was first used (and is still used extensively) in E.coli, and is now used in other prokaryotic and lower eukaryotic organisms as well as higher eukaryotes like rats and mice.  There are several methodologies that are used to accomplish this task  Homologous recombination  Site specific nucleases Sensitivity: Internal  Homologous recombination (HR) (1)  Homologous recombination allows for the introduction of an “engineered mutation” that can be directed to a specific locus within the organisms genome  The process is used to create a loss of function mutation  As of 2009, approximately 11,000 genes have been successfully knocked out of mice. This accounts for roughly half the mouse genome.  HR looks to exploit the cells own DNA repair mechanisms to promote the loss of function of a particular gene. Sensitivity: Internal  Homologous recombination (HR) (2)  Synthetic DNA sequences are introduced to the cells via electroporation or microinjection.  In mammalian cells, the DNA is introduced to the stem cell population  This DNA is usually delivered as a plasmid, containing an insert domain. These domains require a minimum level of homology with the target sequence to integrate into the genome  The minimum required homology is 2kb, however most targeting constructs have somewhere in the region of 6kb to 14kb  During a stem cell experiment, only about 10–2 to10–3 of the DNA integrations are homologous recombination events  The result is gene interference, where an exon is usually substituted for a non-coding region from the Sensitivity:plasmid. Internal Images from Hall et al 2009 doi  Site specific nucleases  There are 4 types of site specific nucleases available commercially for targeted gene knockdown  Engineered homing endonucleases (Meganucleases)  Zinc finger nucleases (ZFNs)  Clustered regularly interspaced palindromic repeats (CRISPR/Cas9)  Transcription activator – like effector nucleases (TALENs)  The repair is initiated either through non-homologous end joining (NHEJ) or homologous re-joining (HrJ). Sensitivity: Internal  Engineered homing endonucleases (EHE)(Meganucleases)  A family of naturally occurring, and incredibly rare cutting, endonucleases.  Divided into 5 families, based on sequence and structural motifs, and are found in all kingdoms of life in some capacity.  Meganuclease I-Sce I, is a homing endonuclease encoded by mitochondria found within bakers yeast (S.cerevisiae).  It recognises TAGGGATAACAGGGTAAT, cleaving at this site producing double Sensitivity: Internal strand breaks Image from Silva et al 2011 doi:10.2174/156652311794520111  Zinc finger nucleases  Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.  They have two main domains, a non-specific Fok1 nuclease domain and a fully customisable DNA binding domain contained between the 3 and 6 individual zinc finger repeats.  ZFNs are able to recognise long target sequences, and promote the removal of a specific sequence from the genome, creating double strand breaks.  The sequence in question is removed from the genome and degraded by local nucleases, with the double strand break being repaired by non homologous end joining (NHEJ). Sensitivity: Internal  TALENS  Seen as an alternative to ZFN’s, and have a similar mechanism of action with a slight variation  DNA binding by these transcription activator-like effectors (TALEs) is mediated by arrays of highly conserved 33–35 amino acid repeats flanked by additional TALE-derived domains at the amino- and carboxy-terminal ends of the array.  TALENs can be generated using a simple “protein- DNA code”, allowing for rapid and effective design of a DNA knockout system. Sensitivity: Internal  CRISPR/Cas9 (1)  CRISPR and CRISPR associated genes (Cas) form an essential part of the adaptive immunity exhibited by some bacteria and archaea, and is a means of them protecting themselves from invading genetic material  Under normal conditions, organisms like S.thermophilus are able to acquire resistance to bacteriophages by integrating a genome fragment from the virus into its genome  Cas9, crRNA (CRISPR RNA), and trRNA (trans- activating crRNA) is required for gene silencing and has become the standard for CRISPR associated gene knockout. Sensitivity: Internal  CRISPR/Cas9 (2)  A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery.  In the absence of a homologous repair template non- homologous end joining can result in indels disrupting the target sequence.  Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.  Indel is a molecular biology term for an insertion or deletion of bases in the genome of an organism Sensitivity: Internal  CRISPR/Cas9 (3) B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair. Sensitivity: Internal  CRISPR/Cas9 (3) C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins. Sensitivity: Internal  Gene knockdown  Technique associated with a change in the expression of one or more of an organisms genes.  The approach can be permeant or temporary.  The gene being knocked down are still expressed by the cell, but the gene product (e.g. protein) levels are significantly reduced compared to the wild type cells. Sensitivity: Internal  RNA interference  A method of transient gene knockdown relying on gene knockdown through mRNA degradation.  RNA interference involves the production of anti-sense RNA to the target mRNA sequence.  There are two main mechanisms of RNA interference, short interfering RNA (siRNA) and short hairpin RNA (shRNA).  Irrespective of the method used, the purpose of the interference is the formation of a RNA induced silencing complex (RISC) and degradation of the target mRNA. Sensitivity: Internal  shRNA vs siRNA Nucleus Generally, shRNA transfection results in a long term gene knockdown (stable), with siRNA transduction usually associated with short term (usually 7 days up to 1 calendar month) or transient transduction Sensitivity: Internal  Over expression systems – Mammalian cells  There are many methodologies that have been identified for the over expression of proteins within lower eukaryotes and prokaryotes, but in cell signalling research we need to focus on higher eukaryotes.  To achieve this we use a wide variety of immortalised cell lines.  In order to have a successful over expression system, you need to have a suitable cell line and a suitable vector. Sensitivity: Internal  Over expression systems  Adenovirus vectors  Medium sized, non-enveloped icosahedral virus  Retroviral vectors  RNA viruses that replicate via a dsDNA intermediate (for example lentivirus’)  Vaccinia vectors  made up of double stranded DNA of nearly 200,000 bp and replicates in the cytoplasm of the host cell  Plasmid based expression vectors Image from Prelich 2012. DOI: https://doi.org/10.1534/genetics.111.136911 Sensitivity: Internal  Adenovirus (1)  Transduce non-dividing and dividing cells  Carry up to 8.5Kbs of heterologous DNA  High levels of transgene expression  Well suited as oncolytic vector  Produced at high viral titres (1010 Plaque forming unites (PFU)/ml) Image used in line with creative commons licence for use within education. Sensitivity: Internal Credit for image: David S. Goodsell, RCSB Protein Data Bank PDB ID: 1vsz / 1qiu  Adenovirus (2)  Highly immunogenic  Vector genome does NOT integrate into host cell genome  Transient expression of the inserted gene sequence  High levels of pre-existing immunity Image used in line with creative commons licence for use within education. Sensitivity: Internal Credit for image: David S. Goodsell, RCSB Protein Data Bank PDB ID: 1vsz / 1qiu  Retrovirus (1)  Exhibit high expression of the transgene of interest  DNA integrates into the host genome and replicates stability  Exhibit low immunogenicity  Produce very stable transformed cells  Low DNA load Image used in line with creative commons licence for use within education. Credit for image: Steven Fuller. Available from Sensitivity: Internal https://wellcomecollection.org/works/ms564a4c  Retrovirus (2)  Can only infect dividing cells  Insertional mutagenesis  Dangerous to handle (Central nervous system diseases, immunodeficiency diseases etc) Image used in line with creative commons licence for use within education. Credit for image: Steven Fuller. Available from Sensitivity: Internal https://wellcomecollection.org/works/ms564a4c  Vaccinia virus (1)  High DNA insert size (25kb)  Expression of transgene is stable.  High Viral titres (3x109 PFU/ml)  Safe to use in most cases (basis of smallpox vaccine)  Breitbach et al 2011 DOI: doi:10.1038/nature10358 Looks at how these viruses can be programmed to target cancer cells Sensitivity: Internal  Vaccinia virus (2)  Can only infect dividing cells  Produces only transient transfection of transgene  Pre – existing immunity can be observed when using these viruses  While safe to use, has been demonstrated to have cytopathic effects (change cell structurally) Sensitivity: Internal  What are the main things to consider when thinking about a gene editing/modification experiment? Sensitivity: Internal