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Methods in Gene Silencing The central dogma of molecular biology describes the flow of genetic information within a biological system: 1. The dogma is a framework for understanding the transfer of sequence information between sequential information-carrying biopolymers, in the most common or general case, in living organisms. There are 3 major classes of such biopolymers: DNA and RNA (both nucleic acids), and protein. 2. The general transfers describe the normal flow of biological information: DNA can be copied to DNA (DNA replication), DNA information can be copied into mRNA (transcription), and proteins can be synthesized using the information in mRNA as a template (translation). Transcription Main article: Transcription (genetics) Transcription is the process by which the information contained in a section of DNA ( gene) is transferred to a newly assembled piece of messenger RNA (mRNA). It is facilitated by RNA polymerase and transcription factors. In eukaryotic cells the primary transcript (premRNA) must be processed further in order to ensure translation. This normally includes a 5' cap, a poly-A tail and splicing. Alternative splicing can also occur, which contributes to the diversity of proteins any single mRNA can produce Genes are regulated at either the transcriptional or posttranscriptional level. So 1. Transcriptional gene silencing (TGS) : is the result of histone modifications, creating an environment of heterochromatin( histone methylation) around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.), Promoters silenced. 2. Post-transcriptional gene silencing ( PTGS): is the result of mRNA of a particular gene being destroyed or blocked. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNAi.( promotor active, gene is hypermethylated in coding region,This resently called RNAi. Other names of post-transcriptional gene silencing (PTGS) : 1. Gene silencing: Scientific Exploitation: Knockouts and Knockdowns epigenetic processes of gene regulation, the ability of exogenous double-stranded RNA (dsRNA) to suppress the expression of the gene which corresponds to the dsRNA sequence., which lead mRNA to unable to make a protein during translation, that occurs without a change in DNA sequence… a. Gene silencing: is naturally triggered when a viral pathogen invades a cell in a plant or animal and enables that plant or animal to stop the virus from replicating and causing disease. b. Although use of this natural mechanism by scientists is still at the early research stage. Gene silencing technology is enabling researchers around the world to protect animals from diseases and develop new crop varieties 2. RNA silencing: refers to a family of gene silencing effects by which the expression of one or more genes is down regulated or entirely suppressed by the introduction of an antisense RNA molecule. The most common and well-studied example is RNA interference, in which endogenously expressed microRNA or exogenously derived small interfering RNA induces the degradation of complementary messenger RNA. It also plays an important role in defending plants against viruses. Enzymes detect double stranded RNA (that is not normally found in cells) and digest it into small pieces that are not able to cause disease. This can be performed under normal conditions or in the context of a disease. 3. RNA interference: is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Or Small Interfering RNAs (siRNAs) are short, double-stranded RNA molecules that can target mRNAs with complementary sequence for degradation via a cellular process termed RNA interference (RNAi) Short history : 1. 1990 Jorgensen : Introduction of transgenes homologous to endogenous genes often resulted in plants with both genes suppressed! *Called Co-suppression **Resulted in degradation of the endogenous and the transgene mRNA 2. 1995 Guo and Kemphues: injection of either antisense or sense RNAs in the germline of C. elegans was equally effective at silencing homologous target genes 3. 1998 Mello and Fire: -extension of above experiments, combination of sense and antisense RNA (= dsRNA) was 10 times more effective than single strand RNA 4.The importance of this technology is reflected by the fact that the 2006 Nobel prize for medicine was awarded for the discovery of RNA interference by Craig Mello and Andrew Fire. What is RNA interference /PTGS? 1. dsRNA needs to be directed against an exon, not an intron in order to be effective. 2. homology of the dsRNA and the target gene/mRNA is required 3. targeted mRNA is lost (degraded) after RNAi 4. the effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism) 5. ssRNA does not work as well as dsRNA General guidelines for target sequence selection include: General Design Guidelines for siRNA 1. Find 21 nt sequences in the target mRNA that begin with an AA dinucleotide. Beginning with the AUG start codon of your transcript, scan for AA dinucleotide sequences. Record each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. 2. Select 2-4 target sequences • Avoid areas with a GC content of >70% or <30%, •Since a 4-6 nucleotide poly(T) tract acts as a termination signal for RNA pol III, avoid stretches of > 4 T's or A's in the target sequence when designing sequences to be expressed from an RNA pol III promoter •Since some regions of mRNA may be either highly structured or bound by regulatory proteins, we generally select siRNA target sites at different positions along the length of the gene sequence. We have not seen any correlation between the position of target sites on the mRNA and siRNA potency. •Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences. 3. Design appropriate controls: A complete siRNA experiment should include a number of controls to ensure the validity of the data. Two of these controls are: •A negative control siRNA with the same nucleotide composition as your siRNA but which lacks significant sequence homology to the genome. To design a negative control siRNA, scramble the nucleotide sequence of the gene-specific siRNA and conduct a search to make sure it lacks homology to any other gene. •Additional siRNA sequences targeting the same mRNA. Perhaps the best way to ensure confidence in RNAi data is to perform experiments, using a single siRNA at a time, with two or more different siRNAs targeting the same gene. Prior to these experiments, each siRNA should be tested to ensure that it reduces target gene expression by comparable levels. Vectors: pSilencer 4.1-CMV Expression Vectors The pSilencer 4.1-CMV vectors employ a powerful CMV promoter to drive high level expression of cloned hairpin siRNA templates in a wide variety of cell types. They also include an antibiotic resistance gene that provides a mechanism to select for transfected cells that express the 1. Mammalian selectable markers The pSilencer 4.1-CMV puro siRNA expression vector contains a puromycin resistance gene to enable antibiotic selection in mammalian cells. Antibiotic selection can be used to enrich for cells that were successfully transfected with pSilencer 4.1-CMV puro by killing off cells that lack the plasmid. Short term antibiotic selection is very useful for experiment systems where low transfection efficiency would otherwise preclude detection of a reduction in target gene expression. For long-term gene knockdown studies, the puromycin resistance gene makes it possible to select cell populations, or clonal cell lines, that stably express the hairpin siRNA. Puromycin in culture medium at 50–4000 ng/mL is commonly used to select cells with an integrated plasmid containing the resistance gene. Cells without the resistance gene are normally killed within 3–5 days. Modified CMV promoter for siRNA expression : The pSilencer 4.1-CMV puro System employs a modified Cytomegalomavirus (CMV) promoter to drive expression with RNA pol II, and includes a modified simian virus-40 (SV40) polyadenylation signal downstream of the siRNA template to terminate transcription.: pSilencer 4.1-CMV plasmids are supplied ligation-ready The pSilencer 4.1-CMV siRNA Expression vectors are linearized with both BamH 1 and Hind III to facilitate directional cloning. They are also purified to remove the digested insert so that it cannot religate with the vector. This greatly increases the percentage of clones bearing the hairpin siRNA template insert after ligation, reducing the time and effort required to screen clones. A basic pSilencer 4.1-CMV puro vector map is shown in Figure 1 on Designing siRNA Hairpins Encoded by siRNA Expression Vectors and siRNA Expression Cassettes These designs produce an RNA transcript that is predicted to fold into a short hairpin siRNA as shown in Figure 1. The selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, For traditional cloning into pSilencer vectors, two DNA oligonucleotides that encode the chosen siRNA sequence are designed for insertion into the vector (Figures 2 and 3). In general, the DNA oligonucleotides consist of a 19-nucleotide sense siRNA sequence linked to its reverse complementary antisense siRNA sequence by a short spacer. Ambion scientists have successfully used a 9-nucleotide spacer (TTCAAGAGA), although other spacers can be designed. 5-6 T's are for cloning into the pSilencer 4.1-CMV vector, nucleotide overhangs containing the Bam H1 and Hind III restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively added to the 3' end of the oligonucleotide. Optimizing Antibiotic Selection Conditions: Cell type, culture medium, growth conditions, and cell metabolic rate can all affect the optimal puromycin concentration for selection of pSilencer 4.1-CMV-transfected cells. Identify the lowest level of puromycin that kills nontransfected cells within approximately 5 days by testing puromycin concentrations from 50–4000 ng/mL while keeping all other culture conditions equal. See step 1. Puromycin titration (kill curve) below. Using the pSilencer 4.1-CMV siRNA Expression Vector 1. a. b. c. Cloning Hairpin siRNA Inserts into pSilencer 4.1-CMV Prepare oligonucleotide Anneal the hairpin siRNA template oligonucleotides Ligate annealed siRNA template insert into the pSilencer 4.1CMV vector d. Transform E. coli with the ligation products e. f. g. h. Expected results Identify clones with the hairpin siRNA insert Pick clones, isolate plasmid DNA, and digest the plasmid with BamHI i. and HindIII to confirm the presence of the ~55 bp siRNA insert. j. Purify pSilencer 4.1-CMV plasmid for transfection pSilencer 4.1-CMV plasmid preparations must be free of salts, proteins, and other contaminants to ensure efficient transfection. 2. Transfecting pSilencer 4.1-CMV into Mammalian Cells: a. Transfect cells and culture 24 hr without selection Transfect the purified plasmid into the desired cell line, plate transfected cells at the plating density and culture for 24 hr without selection It is important to include two non-transfected control cultures. One is subjected to puromycin selection to control for the fraction of cells that survive selection; it will help determine the effectiveness of the transfection and selection. The second control is grown without puromycin selection as a positive control for cell viability. b. Add medium containing puromycin Add culture medium puromycin identified containing the concentration of 3. Selecting Antibiotic-Resistant Transfected Cells: pSilencer 4.1-CMV siRNA expression vectors can be used in transient siRNA expression assays, or to create cell populations or a clonal cell line that stably expresses your siRNA. Note that with normal (nontransformed) and primary cell lines, it may be difficult to obtain clones that stably express siRNA. For these types of cells, it is recommend choosing the antibiotic selection strategies outlined in sections 1 and 2 below a. Short term puromycin selection for enrichment of cells that transiently express the siRNA: Culture the cells for 1–3 days in the puromycin-containing medium, Analyze the population for an expected phenotype and/or the expression of the target gene. b. Selecting a population of cells that stably express the siRNA: Creating a population of cells stably expressing the siRNA involves treating cells with puromycin for several days to eliminate cells that were not transfected. The surviving cell population can then be maintained and assessed for reduction of target gene expression. c. Selecting for clones that stably express the siRNA : the goal is to create a clonal cell line that expresses the hairpin siRNA template introduced with pSilencer 4.1CMV. Cloning stably expressing cell lines is advantageous because strains that exhibit the desired amount of gene knockdown can be identified and maintained, and clones that are puromycin-resistant but which do not express the siRNA can be eliminated.