2011Pranamee Sarmapsarma@hawk.iit.edu[IN CONTEXT TO TREATING MENKES ]Summarised study of 10 technologies that could be used for Gene Therapy, in this assignment thedisease in study is ‘Menkes Disease’ related to poor assimilation and absorption of Copper in the bodydue to mutation or mal-functioning of the ATP7A gene (truncated form is produced).
1) LASER CAPTURE MICRODISSECTION TECHNIQUETitle : Isolation of the seminiferous tubules Application (Gene therapy, e.g Menkes(containing mature spermatids) from the testicular Diesease)tissue (present in the outer edge of this tissue) of STEP 1 : Testes tissues from both sourcea)Menkes affected male b) Normal male to be able (normal and diseased); observe underto isolate the DNA content of the germ cell. microscope where we see the outer edgeIntroduction : Laser Capture Microdissection (LCM) with spermatids and immatureis a low beam laser-assisted method to isolate or spermatozoa (seminiferous tubules)‘dissect’ pure and micro-portions of specific cells STEP 2 : We use LCM to selectivelyfrom a clustered or diverse tissue/cellular system. cut/dissect the outer edge cells from theThe best part being that we can select the specific rest of the tissue by marking the specificpart on the computer screen connected to the LCM regions to be cut, on the computer screen. Laser beam targetted to theseWorking : The laser beam melts the thermo-labile areas cuts and creates visible holes in thefilm attached to stick to the target cells. We get the original tissue.final cells in eppendorf tubes stored beneath thecutting area of the system to collect the sample STEP 3 : The cells selected are collected(there are other ways to select the cells as well). on the caps of the eppendorfs used. TheOnce the area is selected, a tissue capturing seminiferous tubules /sertoli cells areeppendorf tube-cap is placed on the targetted area distinguished and isolated for furtherand a low powered infra-red beam laser cuts the DNA isolation(also RNA) in our casetarget area, the target cells present on the cap are (Centrifugation-sperm pellet-extractionthen transferred to the tube. The tubes placed buffer and proteinase K-centrifugation-underneath (as mentioned earlier) have the target pellet-DNA; add dye or buffertissue cells which can then be taken for further accordingly). We can run the DNAstudies. The main catch of this experiment is obtained on Agarose gel electrophoresis‘tissue-area selection’,’laser beam targetting’ and and observe the band differences (based‘computer monitoring of the process’. A loss at one on dye) and then we can perform DNAof them will produce unsuccessful results. profiling or fingerprinting to study the differences in the normal and diseasedCOST- Approximately 1, 43, 640$ (includes the ( the disease in our case is Xmicroscope, computer software and hardware) chromosome-linked recessive disease)ADVANTAGE DISADVANTAGEInvolves no manual dissection Complex setup, such as computer programs, laser etcFull-proof cell selection and transfer to tubes Targetting large number of cells is tediousTissue retains its morphology, only the specific Some times cells are scattered rather than localisedportion is cut so becomes a problem in fine tuning the targetPure population of cells Full time attention required, process is quite long
2) REVERSE TRANSCRIPTASE – PCR (RT-PCR) TECHNIQUETitle : mRNA from diseased and normal Applications (for gene therapy, e.g in Menkesorganism can be taken and ‘Reverse Disease):transcribed’ to its DNA complement, cDNAfor further studies such as gene therapy. STEP 1 : mRNA is isolated from the technique used above (sperm cell with LCM) usingIntroduction : It is a variant form of normal oligodT beads (pulls out polyA tail and thusPCR (Polymerase Chain reaction) which is mRNA).used to ‘polymerise’ or amplify the STEP 2 : In 2 tubes, the mRNA of normal malerequired sample of nucleic acids. In case of and Diseased male are treated at a specificRT-PCR, there’s an additional step of temperature with Reverse transcriptase‘Reverse transcription’ in the beginning enzyme to give DNA (cDNA) ds copy.where, enzyme ‘reverse transcriptase’transcribes RNA back to DNA and thus the *Through this process we can detect anycDNA is amplified. mutations (e.g Exon skipping or splicing abnormalities, that are most common in Menkes) by comparing the cDNA copy ofWorking : The amplification of cDNA occurs normal to diseased from the mRNA that wein the ‘traditional’ number of 3 steps; FIRST had and thus locate the sites of mutation.: (the main difference than PCR) Reversetranscription of mRNA to DNA (ds) using STEP 3 : Then the targetted cDNA is amplifiedreverse transcriptase at 50⁰C; SECOND : DNA sequencing (Automated DNADouble stranded (ds) DNA melt down and sequencing) with radiolabelled nucleotidesinserted primers anneal along the single from both the samples and then bystranded DNA at 92⁰-95⁰; followed by comparison we can clearly notice theprimer annealing at 65⁰C; THIRD: A special mutation that has occurred (mismatch or(high temperature) enzyme Taq (DNA) nonsense mutation)polymerase extends DNA using the primerstherefore leading to cDNA amplification.Requirements : Thermal cycler, Computer,data and collection equipments, mRNA,PCR special tubes, enzymesCOST : 40, 000$ - 50, 000$ADVANTAGE DISADVANTAGEEarly diagnosis of diseases Need of special primers, thus tediousMeasuring gene expression levels Can pick up contaminants from the
surroundingcDNA production, no introns, thus helpful for Use of two different enzymes, makes it a littlegenetic engineering and modifications between complicated to handle compared to traditionalprokaryotic genes and eukaryotic genes PCR method. 3) QUANTITATIVE POLYMERASE CHAIN REACTIONTitle : qPCR for the above sample is Applications (for Gene therapy e.g Menkesnecessary for quantification and Disease)amplification of the cDNA produced STEP 1 : We use the previous technique’s sample for quantification. RT-PCR is coupledIntroduction : It is a PCR (Polymerase Chain with qPCR for better results.reaction) which is used to amplify andquantify the required sample of nucleic STEP 2 : We label the cDNA from RT-PCR withacids(cDNA in this case). In case ofqPCR, flourescent probes so that through qPCR weflourescent dyes (DNA probes) are used to increase the specificity for our samples tomeasure/quantify the amount of DNA(e.g) enable proper target cDNA quantification forpresent in the starting sample and the final Menkes Disease treatment and analysis.sample. It is basically amplificationdetected by flourescence. STEP 3 : The primers used in the Annealing step are Probed and thus anneal with the specific cDNA product enabling flourescence activity toWorking : The amplification of DNA occurs be observed through the plotting of a graphin 3 steps; FIRST : Denaturation, at 95⁰C; between Flourescence activity against theSECOND : Annealing, at 65⁰C, double number of thermal cycles of PCR. Increase instranded (ds) DNA melt down and the activity will show an increase in the numbersynthesized primers anneal along the single of target cDNA Copies (quantification).stranded DNA ; THIRD: A special (hightemperature) enzyme Taq (DNA) *We can also use this technique to quantifypolymerase extends DNA at 75⁰C using the GENE EXPRESSION when we modify theprimers, leading to cDNA amplification. Promoter sequence for the ATP7A gene of Menkes Disease to observe the pros and consThese temperature important as they are of this therapy (promoter modification)crucial for Primer elongation and DNAhybridisation.COST : 50, 000$ or more depending on theprobes used.ADVANTAGES DISADVANTAGESQuantifying target sample with precision and Costlier than most of the PCR variants due to use
specificity; no primer dimers of specific probes and extra equipmentsBetter method to measure Gene expression Complex with high techincality involvedlevelsReliable method of sample/DNA detection Larger set-up, larger work area 4) DNA Gel Mobility Shift Assay and DNA Footprinting Title : Gel mobility shift assay is to study the Applications (Gene therapy, e.g. Menkes affinity reactions between protein and DNA Disease) : or RNA using DNA footpringting in conjunction STEP 1: We can test the promoter mutation that we plan to do for Menkes diesease (as Introduction : The concept of both the mentioned in the previous page) by this techniques is similar in the fact that both method combination. observe DNA fragments bound with proteins STEP 2: We can mutate the promoter or no proteins. The difference being, Gel sequence(original promoter position can mobility shift assay studies the shifting of a also be determined by this process; RNA band (either up or down) on binding to a polymerase binding to promoter region, -10 protein which can be observed comparing to and -35 sites). Once the promoter is data from DNA Footprinting. mutated we can use this methodology to see if the RNA polymerase along with the other Working : DNA for study is run in different Transcirptional proteins bind to the the lanes, with and without binding proteins. In promoter site (new). The promoter Gel mobility shift assay, we will observe that sequence, if in the proper site (-10 and -35), the DNA bound with protein (heavy) has will be bound by the RNA polymerase and a shifted ‘up’ in the position in lane compared gel run with its fragments, will show a gap in to the same DNA fragment which has no this region compared to original fragment protein bound (therefore, down). In the DNA electrophoresed. To compare the results we footprinting assay, in similar sample we add will have to perform both DNA Footprinting Dnase I which cleaves protein unbound DNA (for the new promoter sequence) along with at random sites and leaves the protein-bound the Gel mobility shift assay (for the RNA DNA intact. The gel run with these samples, polymerase + Proteins bound to the new can be autoradigraphed (using DNA probes in promoter). the samples) and the difference in a conitnuous cleaved Lane and protein-bound STEP 3: If successful, we can use this process DNA protected lane can be observed by the in up regulating theATP7A gene transcription dissimilar patterns and a missing portion of for treatment (gene therapy) of Menkes DNA (GAP, due to proteins bound). Disease COST : Electrophoresis – 1600$ approx; General accessories – 500$, Chemicals- 100$
ADVANTAGES DISADVANTAGES Precise method to study interactions Special conditions need to be maintained for high efficiency Reveal protein and nucleic acid interaction Not all protein-nucleic acid interactions are complexes solved Low cost. Technically tedious 5) SDS-GEL and 2D GEL ELECTROPHORESISTitle : Gel electrophoresis of biomolecules to Applications (gene therapy, e.g Menkes Disease) :separate them on the basis of size/chargewhere network of agarose molecules(gel) STEP 1 : We got proteins from both of the normalserve as a sieving medium and diseased Males. We need to test them for their properties with the original protein (ATP7AIntroduction : Biomolecules such as Proteins gene). We take one sample of protein from eachand nucleic acids have to be separated which and run in a gel in different lanes. E.g.is done by Electrophoresis of the sample (with LANE 1 2 3 4 5a dye/buffer) along a gradient (pre-decided)on an agarose gel with different conditions. Incase of SDS-Gel (PAGE;separates proteins), aspecial component called Sodium DodecylSulfate (SDS) is added to the separating gelpart while preparation. In case of 2D Gelelectrophoresis, the separation if by 2directions (1 D) and then the other direction isperpendicular to the previous run.Working : In the SDS-PAGE technique themain catch is addition of SDS ( a detergent) in STEP 2: We can thus combine 1D as SDS and 2Dan optimum amount so that the SDS for a different property and get a result similar todenatures the protein molecules making it above. E.g as we can se, protein from normalnegative (constant charge for all), thus final male gives us two bands compared to theseparation is on the basis of Mass. In the 2D molecular weight of the protein marker band, aGel electrophoresis, the biomolecules, e.g similar band is seen with the ATP7A fromProteins are first separated on 1D i.e on one promoter mutant 1, but not from the diseased orproperty. The 2D continues from there for the ATP7A from promoter mutant 2 form. Thusseparation on a second property. The basic for therapy then we can use the promoter mutantprinciple for this system lies on the fact that 1 form.no two protein molecules (in this case) will besimilar in 2 properties. So if not separated *We can thus validate our therapy with suchthrough 1D they are bound to do so in 2D comparisons.COST : Chamber and kit = 1600$ approx;Accessories and chemicals = 600$ approx
ADVANTAGES DISADVANTAGESConvenient visual method of molecule detection Agarose gels are temperature sensitive, esp. HeatBetter resolution than the rest, due to dyes Some dyes (EtBr, e.g) are harmful for humansFast process ( 1-2 hour run) Quantification of molecule is approximateEasy to set up Hard time preparing solutions with exact concentration 6) Cell Culture System Title : Culturing or growing of cells isolated by Applications (gene therapy; e.g Menkes LCM/etc under special conditions and Disease; Sperm cell isolated using LCM medium to give a cell mass. technique) : STEP 1 : Spermatozoa from the sertoli cells Introduction : We isolate cells to study, but of testes can be grown as cell culture. From for this they need to be maintained in vitro the primary cell line, cells taken can be with conditions suitable for their growth and injected with the new gene or new somewhat similar to their in vivo conditions. enhanced promoter mutant screened from The cells grown into a mass are then referred above techniques into the cell. These cells to as a Cell culture system e.g Primary cells or can then be cultured on medium such as secondary cells. These can then be stored and those for Embyronic stem cells (closest preserved (immortal cell lines) for study in the match), etc with addition of serum proteins future, especiall for human diseases and and stimulating hormones. benefit. STEP 2 : Temperature is maintained at the Working : Cell cultures can be prepared by body limit of 37⁰C in the growth incubator creating special in vitro conditions with and cell kept in the medium for a time various machines, chemicals and period. technologies. A cell is isolated from a specific part of an organism (Study organism) and STEP 3 : The growth media should be then grown on a nutrient medium, placed maintained and refreshed time to time for under an incubator (growth chamber,etc) for a better productivity. The cells can be a specific period of time. The cell culture either grown on a small petri dish or a large obtained directly from the first isolation and flask. cell growth is called Primary cell Line. A Secondary cell Line is produced from a cell *We can observe all the changes and the culture grown from cells taken from the manipulations in this system itself, which Primary Cell Line. The secondary cell line can mimics in vivo conditions in the laboratory. be maintained for a longer duration compared to the Primary line. COST : Glasswares cost 200$ approx; Equipments and lab set up approx 100, 000$
ADVANTAGES DISADVANTAGES Production of human vaccines and drugs Contamination problems of cell lines Helpful in Gene therapy Tedious maintenance of cell lines Growing of organisms, such as plants, in vitro Chances of contact inhibition in the system for cells growing Extensively used in protein study and production High cost 7) Western Blot TechniqueTitle : To determine the size of a protein or Applications (gene therapy, e.g Menkesquantify protein expression Disease) :Introduction : Western Blot technique is a small STEP 1 : We can use this method to detect ourdiversion from the usual Southern and Northern new-promoter-induced proteins and theirblot techniques. It basicall works on the ligand- interaction with the standard antibodies (insubstrate binding wherein specifically protein ase of normal). Solubilise the proteins (normalbinding to antibodies or etc, is made use of. The and dieseased) in a suitable buffer and preparetechnique uses both gel electrophoresis for SDS-PAGE. Once the gel, as obtained(normally, SDS-PAGE) and Western blotting above, is produced, we stack it over a pile of(basically transferring the gel-run bands onto a filter papers, with a nitrocellulose membraneNitrocellulose membrane) in between and kept in a buffer tray. (Cathode and Anode transfer method can also be used).Working : The SDS-PAGE seperates the proteinson the basis of their molecular weight. This STEP 2 : The induced and uninduced (gene)seperation is also based on the concentration of proteins bands are transferred onto thethe acrylamide gel. The gel thus obtained is then membrane in the same pattern as in the gel.‘blotted’ onto a membrane (nitrocellulose), This membrane is then treated with buffer,transferred from cathode to anode. Once the antibody solution, washed again for freeproteins (bands)are transferred from gel to the antibodies. The antibody specific to the targetmembrane, the membrane is then probed with protein binds to the band area with thattarget protein-specific antibodies which have protein (normal and induced) and in turnthe property of (chemical reaction, colorimetric) produces a chemical luminiscence which canchemiluminescence based on the activity of be detected. The membrane is exposed to X-antibody-protein binding detected by an ray film for about 1 hour, in a dark room.detector which records this activity and thus Processed and visualised in the dark room,aids in quantifying protein activity. showing luminescence (bright ; white areas) where the antibody is bound to the protein.COST : Kit (containing buffers, antibodies, This way we can validate our new modifiedenzyme conjugates costs 300$. Electrophoresis protein (ATP7A) which is almost similar to theprocess – 2000$ approx, Membranes – (15 normal proteins (antibody binding property).sheets) 107$, Detection kits – 323$, X-Ray film-70$
ADVANTAGES DISADVANTAGES Easy method to detect Protein amount and Complicated set-up expression Helps in detecting immunogenic reactions Cost factor arises due to X-ray filming and different kits that are used High level of sensitivity and precision Needs extremely careful handwork while transferring of bands from gel to membrane Detects even minute protein activities Time consuming method 8) Mass Spectrometry for Protein AnalysisTitle : To characterize and identify proteins using Applications (gene therapy, e.g. MenkesMass Spectrometry (MS) disease) :Introduction : The MS identifies the STEP 1 : We have protein samples fromcharacteristics of the proteins (broken into normal and diseased males. We can use MS topeptides) which can then be studied comparing determine the characteristics of there twoto established protein databases. They basically different protein (ATP7A) samples to mark theionise the molecule of study (protein) so that difference and also to understand theirthese can be manipulated under the magnetic fragment organisation plus the sequences.fields of the system to determine the STEP 2 : We take the samples one by one, notm/z(mass/charge) ratio. (* Magnetic fields can together to avoid complexity in resultonly workd on charged ions; cations in this case). interpretations.Working : The MS is basically divided into three STEP 3 : For instance Protein N (from normal)parts -1) Ion Source (ESI, Electrospray ionisation, is ionised into N⁺ (z=1), the graph observedor MALDI, Matrix-associated laser desorption or will have the first peak showing N⁺ (original)ionisation, for proteins); 2) Mass Analyzer; 3) and then subsequently the fragments of NDetector. The protein sample is prepared for the would show up which can then be comparedfirst step (ionisation). The sample is hit by with the database and the Diseased sample.electrons giving rise to a radical (cation) by losing Protein D (from diseased) will be similarlyelectrons. The charge of this ion is usually 1, thus ionised into D⁺ and then graph with D⁺ as them/z ratio generally helps us determine the ‘Mass’. first peak (molecular weight can beDuring the process, the original sample gets determined by this).fragmented due the peptidase activites. The Massanalyzer basically focusses different ions of STEP 4 : We can compare the data from bothdifferent mass (fragments) onto the detector, and then design a scheme for a geneticallydirected by the magnetic field. When an ion hits modified protein, with natural propertiesthe detector screen, a signal is recorded which is (including the structure).then plotted in a graph through the computer. *Through this method we can also determineThis is compared to the established database ofthe sequence of the protein and related fragment - ATP7A protein interactionssequence is then determined. This also gives way - ATP7A pre-mature and mature form; Theseto the molecular structure of the protein. will further help us in our gene therapy.COST : 80,000$ - 100,000$ (depends on the type)
ADVANTAGES DISADVANTAGESInsoluble samples can also be used Volatilisation of samples is a tough stepNon-polar samples can also be used Thus, difficult with non-volatile compoundsProtein characterisation, mass and structure Diffuculte to clean due to set up, thereforedetection is possible chances of contamination possibleSmall sample size is also good High maintenance costs 9) DNA Microarray (Chip technology in biology) Title : To study and detect different levels of gene Applications (gene therapy, e.g Menkes expression in organisms using microarray (chip) Disease) : system STEP 1 : This time use this technology to Introduction : DNA Microarray is a small chip with check if our mutant ATP7A producing organised rows of specified size allowing DNA truncated gene is due to exon skipping of (cDNA) to hybridise and a scanner to detect the the gene. activity of subsequent gene profiling experiments STEP 2 : Use mRNA from both normal and on the chip. It is a convenient method observe diseased males; reverse transcribe them gene expressions, genotypes, mutant genotypes. into cDNA; amplify cDNA using PCR; radio label them green (normal) and red Working : The Microarray is basically a solid chip, (diseased) for easy detection. The colour with attachement fabrications for cDNA. We say Yellow would mean ‘normal and diseased cDNA because gene expression can be detemined share the same pattern there’. We should only by studying mRNA (but DNA represents a have prior knowledge of the standard gene). We, therefore, isolate mRNA and use exons. Following incubation of DNA on chip enzyme Reverse Transcriptase (mixed in the with cDNA (labelled), we observe buffer for radiolabelling of DNA) which transcribes something like this : mRNA back to cDNA copy. This is then PCR amplified for a huge number of copies and placed on the DNA strands (single) on the microchip allowing them to hybridise (base-pairing with complementary strands). This we the hybridised strand emit the radioactivity through which we can detect the expression levels, expressing genes and also look for ways for gene therapy. COST : Chip costs about 50$ to 2500$. The entire set –up would be around 100-200, 000$ *We can then insert proper exons once we know the locations and sequences using computer tools and previous databases.
ADVANTAGES DISADVANTAGESLots of information in single go Highly Attentive skills requiredWe can study gene typing in one experiment Micro arrays need to be fabricated everytime according to the needs of the experimentSmall chip, therefore no storage problems Might have hybridization problemsSmall amounts of sample required 10) PRODUCTION OF Knock Out Mice (abnormal mice) Title : A gene is knocked out in mice to study its Applications (gene therapy, e.g. Menkes effect and importance compared to normal Disease) : To study importance of ATP7A gene progeny. STEP 1 : Isolate the gene ATP7A from the Introduction : A gene (in DNA) can either be on cDNA library (actual gene, no introns). The or off (naturally), but if a gene is artificially gene is then disrupted so that it is rendered ‘switched off’ or inactivated or disrupted helps non-functional or mal-functional (can be done in studying genotype and phenotype. by inserting second gene, e.g antibiotic resistane that mouse usually lack, into the Working : A gene in study is isolated (can be also essential coding region). This construct can from cDNA library), disrupted off (knocked-out then be inserted into a Vector that is then gene) its normal function, cloned into a suitable injected into the Embryonic stem cells vector that can be transferred to the mouse- (Blastocyst). A new mouse progeny can be stem cells (present in the blastocyst stage of detected by certain phenotypic changes mouse embryo) or directly to fertilised eggs, contrast to normal mouse. The affected implanted in the mouse and allowed to form mouse will have kinky hair, pigmentation, progeny. The changes that are observed in the skeletal defects, etc relating to Menkes new born progeny (50-50 chances, dominant disease. gene and recessive gene) are carefully observed against the progeny where the particular gene is STEP 2 : still functional. Through such methodology we Mouse get to know of a gene function and the changes Blastocyst that occur due to a change/error in the gene. The mouse containing the knocked-out gene product is known as a knock-out mouse and its Mouse 1 progeny (knock-out gene dominant) too would have the same features (if the parent mouse survives). COST : Producing such knockout mice costs 5, 000$ - 30, 000$ (Mouse, reagents, instruments, kits, vectors and breeding)
Some have kinky hair, small structureADVANTAGES DISADVANTAGESStudy of gene functions Some features generally differ from humans, not successful everytimeStudy of gene struture and regulating elements Producing such a mouse is expensive (also breeding)Study of mutations Maintenance of transgenic mice needs more skill and staffDrug development for various diseases User has chances of contamination and thus diseased