Abstract: Epigenetics is a mechanism functioning above the level of the gene such that the behaviour of the same gene is altered, enabling it be differently expressed in different cells (Conrad H. Waddington, 2012). Epigenetics now involve a wide range of alterations resulting in DNA methylation, histone post-translational modifications, and alterations in nucleosome positioning (Hwang et al., 2017). DNA methylation is catalysed by enzymes known as DNA methyltransferases (DNMTs) and involves the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the 5-position carbon in the DNA base cytosine to produce 5-methylcytosine. DNA methylation is exemplified by the silencing transcription taking place at CpG islands in gene promoter regions. This system has been modified to take advantage of the specificity of the guidance RNA and at the same time, by fusing the methylase gene to the Cas9, achieve methylation of the targeted gene. The initiation of tumours is strongly dependent upon comprehensive DNA hypo-methylation, which affects a range of genomic sequences such as CpG poor promoters, gene deserts, introns, repetitive elements and retrotransposons. The mitochondria as double-membraned organelles concerned mainly with adenosine triphosphate (ATP) production along with several other key cellular functions. The emergence of genome manipulation tools has strengthened the investigation of epigenetic processes in both prokaryotic and mammalian eukaryotic cells by facilitating widespread use of microbiological tools such as the CRISPR-Cas9 system, zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALENs).The aims of this project , therefore, includes: generation and validation of a genetic tool to manipulate the epigenetics of DAN mitochondrion POLG gene, to perform the sequences of sgRNA will be cloned and validation specific sequences for the Polymerase gamma (POLG). Methylation of DNA mitochondrial POLG gene will be done by using a modified CRISPR-CAS9 system. Then, tissue culture transfection of mammalian cells with dCAS9-methylase plasmid system into mammalian cells, DNA isolation and assessment of the methylations by qPCR amplification and confirmation of altered gene expression via western blot. The information gathered from this study will be useful for the understanding the molecular mechanism of various disease like cancer.
1- Introduction and Background: 1-1 Epigenetic: Epigenetics is a mechanism functioning above the level of the gene such that the behaviour of the same gene is altered, enabling it be differently expressed in different cells Conrad H. Waddington (2012). The development of an organism depends upon patterns of gene expression which are regulated both in time and space. This is achieved partly by so-called epigenetic changes – changes to the DNA and associated proteins without altering the DNA sequence. Within the genomes of the eukaryotes, hereditary information is communicated by chemical alterations of both the DNA and chromatin-associated proteins. The chromatin structure allows the genetic information encoded in DNA to be packed within the cell in a tight and exact structure which powerfully controls the capacity of the genes to be activated or inactivated. Thus, by regulating chromatin structure and DNA accessibility, epigenetic changes have significant effects upon gene expression. The sum of these epigenetic alterations, at the level of the genome, is termed the epigenome. Razin (1998) describes the epigenome as consisting of the chromatin and its alterations along with a covalent alteration (methylation) of the cytosine rings at the dinucleotide sequence CG. According to O’Carroll et al. (2001), these epigenetic processes are powerfully adaptive according to the cell type and the stage of development and are thus instrumental in comprehensive cell-specific sequences of gene expression. Meaney and Szyf (2005) have described the manner in which various cell types carry out specific gene expression schemes that are powerfully sensitive to environmental, developmental, physiological and pathological signals. Thus, epigenetics is the study of the heritable and reversible control of attributes that does not depend upon the DNA succession. According to the published literature, there are two main categories of epigenetic alteration, each of which have deeply significant impacts upon gene regulation, development, and carcinogenesis. These are: (I) DNA methylation, i.e. chemical modification of the cytosine residues of the DNA, and (II) histone modifications – changes in the proteins associated with DNA (Anon, 2017). According to Egger et al. (2004), recent studies have shown that, due to the central role of epigenetic modifications in the schedule of gene expression, epigenetics exerts a key influence in a wide range of illnesses. While cancer is the disease most commonly linked to abnormal epigenetic processes, Sarkar et al. (2014) have pointed to recent evidence suggesting a role in cardiovascular, metabolic and neurological conditions. Furthermore, according to Hwang et al. (2017), the concept of the reversibility of epigenetic changes has led experts to suggest that the restriction of epigenetic changes may have major curative possibilities. Epigenetic modifications of gene expression are more sweeping than the genetic changes that can lead to cancer, frequently affecting more than one gene. Two categories of anti-carcinogenic drug have been demonstrated to counteract the aberrant epigenetic alterations that lead to cancer, namely: (i) DNA methylation inhibitors and (ii) HDAC inhibitors. Epigenetic medications are also presently being employed for the treatment of certain neurological conditions and malignancies.According to Hwang et al. (2017) the field of epigenetics now encompasses a wide variety of alterations leading to methylation of the DNA and post-translational changes to histone, as well as phosphorylation, addition of ubiquitin to a protein (ubiquitination), acetylation and alterations to nucleosome arrangement. These alterations are passed on from one cell generation to the next by mitosis and from one organism generation to the next by meiosis (Esteller, 2011). Jones and Baylin (2007) argue that the developmental aspects by which silencing is achieved, along with the resulting high level of mitotic stability, means that the pathological silencing of genes that control growth, as well as other genes, is a central aspect of human development.The majority of chronic illnesses are significantly influenced by lifestyle choices. In particular, an unhealthy diet or inadequate nutrition can disrupt the normal behaviour of the epigenome, leading to the onset of various chronic diseases, including cancer. On the other hand, Link, Balaguer and Goel (2010) argue that the risk of cancer can be reduced by the observation of a suitable fiet including dairy produce. According to Poirier and Vlasova (2002), the diet has an effect upon both hypo- and hyper-methylation, influencing the methyl-metabolism pathway in the first case and targeting DNMT expression and activity in the second. A diet lacking in choline, folate, methionine, or selenium, for example (methyl deficient) can influence DNA methylation and lead to metal/mineral-associated conditions including atherosclerosis, birth defects, cancer, neurological disorders or pancreatic lesions.
1-2 DNA methylation:The most thoroughly examined and understood type of epigenetic change involved in human cancer is that of DNA methylation. According to Hwang et al. (2017), DNA methylation is catalysed by enzymes known as DNA methyltransferases (DNMTs) and involves the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the 5-position carbon in the DNA base cytosine to produce 5-methylcytosine (see Figure 1). This can trigger mitotic proliferation of the altered arrangement with consequences for the influence of administrative proteins such as interpretation factors (Anon, 2016). This process occurs in a wide variety of organisms, including both prokaryotes and eukaryotes, and generally results in gene suppression. According to Wilson and Murray (1991), DNA methylation in prokaryotes occurs on both cytosine and adenine bases and constitues part of the host restriction system. According to Bestor (2000), five types of DNMT, namely DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L, have been linked to the DNA methylation process in mammals.DNA Methylation has become recognised as a primary mechanism controlling the cellular reprogramming processes including cell differentiation, cellular ageing, and disorder. Klose and Bird (2006) have described the three main enzymes concerned with setting up and sustaining DNA methylation sequences. The sequence is initiated by the methyltransferases DNMT 3A and 3B and is sustained by DNMT1, which guarantees that the methylation sequences are accurately copied during each cell division. According to Jones (2012), DNA methylation in mammalian cells takes place primarily on the cytosine residues adjacent to guanine (so-called cytosine-guanine dinucleotides, or CpGs) and is often linked to gene repression when it occurs at enhancers and promoters.DNA methylation is exemplified by the silencing transcription taking place at CpG islands in gene promoter regions. Studies employing high-throughput methylome datasets have implied that DNA methylation appears to modulate gene expression patterns in different ways by occurring both within gene-coding (intragenic) regions and between gene-coding (intergenic) regions. For example, increased levels of DNA methylation have recently been observed at exon 2 of PolgA in mouse ESCs during differentiation into neurones. While high levels of DNA methylation were associated with low mtDNA copy number and low POLGA expression in pluripotent and multipotent cells, decreased levels of DNA methylation at exon 2 of POLGA were associated with higher mtDNA copy number in terminally differentiated astrocytes. Notably, cancer cell lines such as the glioblastoma multiforme HSR-GBM1 cells were unable to demethylate exon 2 of POLGA and failed to differentiate (Lee et al., 2015). According to Neidhear (2016), DNA demethylation can be either latent (occurring without methylation of recently integrated DNA strands by DNMT1 during a small number of replication rounds, e.g. upon treatment with 5-Azacytidine) or dynamic (occurring via direct methyl expulsion independently of DNA replication).
Figure 1: DNA methylation and Demethylation process (Hwang, 2017) Gene activation a repression is indicated by DNA methylation and hydroxymethylation, respectively (Bird, 2002). The impacts of these mechanisms stretch across extensive chromosomal regions to create memorised gene expression states. Although DNA methylation was previously considered to be irreversible, Miller (2007) has pointed to recent evidence that demethylation can occur, the conversion of 5-methylcytosine to 5-hydroxymethylcytosine being catalysed by a newly recognised group of dioxygenases known as the ten-eleven translocation proteins TET1, TET2 and TET3 (16, 100). The hydroxymethyl group is reactive and can rapidly regenerate un-methylated cytosine (dashed arrow in Figure 1), leading to gene activation.3-1 Post Translational modifications of Histone:The nuclear DNA in eukaryotic cells is not isolated, but occurs in association with numerous proteins to form a chromatin complex. The chief proteins in chromatin are the histones – alkaline proteins which are accountable for the immense degree of packaging that confines the DNA within the eukaryotic nucleus. The basic chromatin unit is the nucleosome, which is described by Rosa and Shaw (2013) and Mariño-Ramírez et al. (2005) as being built up from 147 base pairs of DNA wrapped around a histone octamer consisting of two copies of histones 2A, 2B, 3 and 4. Hwang et al. (2017) describe the structure of the histone proteins as consisting of a core region with the sites for post-translational alterations such as acylation, methylation, phosphorylation, poly (ADP) ribosylation, sumoylation and ubiquitylation being supported by unstructured tails at the amino end of the protein.Methylation of DNA can modify the chromatin to generate a more compact structure which results in suppression of gene expression. Epigenetic mechanisms such as chromatin re-modelling and histone modifications specifically involving the N-terminal tails, have recently been found to work together to regulate chromatin structure and, hence, cellular mechanisms including gene expression and even DNA methylation. Although research into histone tail modifications has focused primarily upon acetylation and phosphorylation, other modifications to H3 and H4, such as methylation and ubiquitination, have recently been shown to be central to the behaviour of their associated chromatin.Histone methylation primarily involves transfer of a methyl group from the active enzymatic donor SAM to the e-amino groups of lysine, serine or arginine residues within the H3 and H4 tails, catalysed by a family of enzymes termed histone methyltransferases (HMTase). Methylation is more complex than acetylation and, according to Lachner and Jenuwein (2002), supplies an extra level of control over gene expression by inducing changes to chromatin structure. According to Volkel and Angrand (2007), each methyl group adds 14 Daltons to the histone protein and effects the folding of chromatin by an electrostatic mechanism. Tamaru and Selker (2001) have observed a reduction in genomic DNA methylation when wild type Neurospora histone H3 lysine 9 is replaced by H3 with an altered amino acid at position 9, which is resistant to methylation.
Figure 2: Histone tail of modification H3. The DNA in the eukaryotic cell is wrapped and packaged in chromatin complexes. The principal unit of chromatin is the nucleosome, which consists of histone octomers comprising two each of H2A, H2B, H3 and H4 in( figure 2). The DNA wraps tightly around the nucleosome and combines 8 histones into the chromosomes. According to Triantaphyllopoulos, Ikonomopoulos and Bannister (2017), the histone within this complex experience numerous covalent post-translational modifications (PTMs) which can potentially carry epigenetic information and can impact upon gene expression by altering chromatin construction and selecting histone transformers. Histone alterations affect a range of natural functions including initiation or deactiviation of transcription, chromosome movements and DNA repair. In a large number of species, histone H3 acetylation occurs primarily at lysine number 9, 14, 18, 56 and 23, methylation occurs at lysine number 4, 9, 27, 36 and 79, and at arginine number 2, while phosphorylation occurs at serine numbers 10 and 28, and at threonine numbers 3 and 11. By contrast, histone H4 is primarily acetylated at lysine numbers 5, 8, 12 and 16, methylated at lysine number 20 and arginine number 3 and phosphorylated at serine number 1.5-1 Diseases and epigenetics: Although epigenetic modifications are essential for normal development and health, they can also be involved in governing disordered conditions. Disruptions to any of the three systems that contribute to epigenetic processes can lead to abnormal initiation or inhibition of factors, leading to disordered growth, neurodegenerative illnesses, cancer and other mitochondrial disorders. 5-1-2 Cancer:The onset and development of cancer involve pronounced alterations in DNA methylation which were the first epigenetic changes to be recognised in the disease Feinberg and Vogelstein (1983). Rodriguez et al. (2006) noted that the initiation of tumours is strongly dependent upon comprehensive DNA hypo-methylation, which affects a range of genomic sequences such as CpG poor promoters, gene deserts, introns, repetitive elements and retrotransposons. Previous investigations have highlighted the combination of gene-specific promoter hyper-methylation and comprehensive genomic DNA hypo-methylation as an epigenetic feature of cancer cells. Extensive DNA methylation had been observed in the pluri-potent cells in conjunction with low POLGA and low levels of mitochondrial DNA. According to Lee et al. (2017), the methylation of PolgA exon 2 has been shown to decrease as the copy quantity of mt-DNA increases in irreversibly isolated astrocytes. Surprisingly, tumour cells similar to the glioblastoma multiforme HSR-GBM1 cells that fail to isolate remained unable to de-methylate the PolgA exon 2. DNA Overall, exon 2 DNA methylation was optimally reduced in class cell partition and mt-DNA replication. The outcome of this study determined the limit of DNA methylation into the sequence of a replica amount of mtDNA, which is a fundamental determinant for cell detachment as well as affecting the choice of developmental cell modifications and methylation of PolgA exon 2 and influencing the onset of tumour cell formation. Furthermore, mitigation of the tumours is associated with reconstruction of the primary print amount of mtDNA, suggesting that the mt-DNA copy number is linked to an inherent fixed cellular incentive.6-1 Mitochondria POLG and Diseases: The mitochondria as double-membraned organelles concerned mainly with adenosine triphosphate (ATP) production along with a number of other key cellular functions (Sun and St. John, 2016). According to Chan and Copeland (2009), there exist within each mitochondrion numerous copies of the circular double-stranded mitochondrial genome (mtDNA), the structure of which is built up from 16, 569 base pairs and which encodes the essential subunits of the electron transfer chain (ETC). The ETC involves five complexes which take part in ATP production via the oxidative phosphorylation (OXPHOS) pathway. According to DiMauro and Schon (2003), ATP production via mitochondrial oxidative phosphorylation is centrally reliant upon the structural soundness of the mtDNA, which encodes 13 key polypeptide subunits of the respiratory chain, which integrate with more than 70 nuclear subunits to create the ultimate standard pathway for energy metabolism. Stumpf and Copeland (2010) have stressed the central role of the mitochondria in generating the preponderance of ATP in eukaryotic cells. Numerous studies in recent years have demonstrated the damage arising from mutations in the mtDNA potentially disrupting ATP production and leading to disordered cellular function or cell death. Isolated mutations in the nuclear DNA (nDNA), or aberrant expression of the mtDNA transcription and replication factors encoded in the nDNA, result in impaired mtDNA transcription and replication, which may lead to diminished oxidative capacity and energy generation. It has been demonstrated that mutations in POLG can result in a shortage of mtDNA and stimulate breast cancer, with clinical symptoms often appearing as soon as these deficiencies affect the cellular metabolism. While mutation in the mtDNA sequence is the primary source of human mitochondrial disease, Hudson (2006) has cited several recent studies implying that mitochondrial disease can also arise from mutations of the gene encoding POLG, since this impacts upon the repair or replication functions of the mtDNA. Alpers-Huttenlocher syndrome and progressive external ophthalmoplegia, for example, are linked to POLG impairment. Methylation of the POLG gene in the mitochondria of a variety of cells can lead to various pathologies. For example, neurodegenerative diseases such as Alzheimer’s disease are linked to methylation of the POLG gene in the mitochondria of the neurones. The investigation will smear the damaged CRISPR CAS9 structure to examine the methylation of the POLG gene, the level of methylation being assessed via limitation assimilation chemical and qPCR. Figure 3: mt-DNA replication and interpretation occur inside the mitochondria. the replication of mtDNA is dependent upon replication factors which are encoded in the nDNA and translocate into the mitochondria in figure 3 . These include the mitochondrial single-stranded-restricting protein (mtSSB), Twinkle, POLGA, POLG adornment subunit (POLGB), and DNA topoisomerase I mitochondrial (TOP1MT). A collection of upstream controllers such as STAT3, HIF1α, SIRT1, MYC, PGC1α/β, NRF1/2, ERRα/β/γ, SIRT3, and PPARα/β are involved in modulating articulation of the factors directly controlling the interpretation and replication mtDNA (Sun and St. John, 2016)
7-1 dCAS9 Methylase, a tool for specific gene methylation:The emergence of genome manipulation tools has strengthened the investigation of epigenetic processes in both prokaryotic and mammalian eukaryotic cells by facilitating widespread use of microbiological tools such as the CRISPR-Cas9 system, zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALENs). Ishino et al. (1987) describe clustered regularly interspaced short palindromic repeats (CRISPR) as 29-nucleotide repeat sequences separated by various 32 nucleotide spacer sequences. These were first observed in bacteria in 1987 and are associated with the nuclease protein Cas9 (CRISPR associated protein 9). In bacteria and other single-celled organisms, the CRISPR/CAS9 system acts as a highly complex type of immune response which helps to defend the organism against contamination by foreign DNA. Invading DNA is first recognised as foreign, then scraps of the foreign DNA sequences are collected and incorporated into a genome locus which is then transcribed to produce first a pre-CRISPR RNA and finally the full crRNA.According to Doyle et al. (2013), genome editing tools based on this system have found widespread use in the targeted modification of any chosen gene sequence, so that epigenome editing now facilitates the concerted investigation into the relevance and applicability of specific epigenetic modifications at a given locus or a genomic region. The basic arrangement of an epigenetic editing tool incorporates two components, namely a DNA-restricting area and a nuclease space. While domains can be targeted on the basis of ZFNs, Stories or the CRISPR-Cas9 system, the primary approach is to use ZFNs. The functional region of the epigenome editing tool achieves the desired epigenetic modification at the targeted domain. According to Vojta (2016), two essential epigenetic systems for quality modulation are post-translational modifications of histone tails and adjustments of DNA particles by cytosine methylation, each of which present attractive targets for control. According to Carlson et al. (2012), these methodologies can be used to alter the genome of a cell either in culture or in viva. Although the majority of the long-established techniques for genome analysis are still indispensable for the elucidation of fundamental cell function, the intricacies of gene regulation have always been inaccessible to these methods. According to Falahi et al. (2013), efforts to redress the shortcomings of these established genome analysis methodologies, the genome editing tools have been adapted to facilitate targeted alterations such as acetylation, methylation, de-methylation, cytosine methylation and hydroxymethylation of histone tails.The development of epigenome editing tools has made it possible to study the effect of targeting a specific location for modification of a specific region of the genome. Sarkar (2014) have cited investigations aimed at providing an understanding of how the epigenetic characteristics of abnormal cells might be reversed, hence providing potential treatments. The drawback of this approach is that it requires a high level of scrupulousness and needs to comprehensively address all aspects of the condition. According to F (2017), epigenetic modification has therefore taken on a central role in separation of developing microorganisms, design of medication design, and quality treatment. Cong (2013) reports that studies of epigenetic modifications have predominantly employed the CRISPR Cas9 system because of its natural ability to target a variety of regions on the genome under the direction of guide RNA. The CRISPR-Cas9 system can target any 20 bp genomic DNA sequence which is immediately followed by the 5′-NGG-3′ protospacer adjacent motif (PAM). In the human genome, this generally occurs every 8 – 12 bp.Sections of prokaryotic DNA consisting of diminutive, surplus base sequences are known as bunched routinely interspaced small palindromic rehashes. CRISPR Cas9, a vital modification of the CRISPT Cas9 configuration, has been adapted to edit genomes. The Cas9 nuclease complexes with prepared guide RNA are inserted into the cell, making it possible to excise specific regions of the cell’s genome and, hence, remove existing characteristics and insert new ones. The functional element of the CRISPR CAS9 system is the nuclease substance which makes site-coordinated alterations to the genome. The post-translational modification of histone tails and the modification of DNA by cytosine methylation are basic epigenetic techniques for the regulation of characteristics. This study will be linked to an altered version of the editing system to improve methylation. The aims of the project to characterize and validation of a genetic tool to manipulate the epigenetics regulation of the polymerase gamma gene by persuade hyper methylation in the CpG islands of the promoter of Polg gene using DAMT3A- DCAS9 and using dcas9- pgRNA genetics tool. In order to determine clone and validate specific pgRNA sequences for targeting the specific gene which is Polymerase Gamma.
2- Material and Methods:LentiGuide- puro (Addgene plasmid Catalogue no 52936) and PgRNA (Addgene plasmid Catalogue no 84477) was purchase from Addgene. T4 ligase Buffer (New England BioLabs , catalogue No B0202S) , T4 Polynucleotide Kinase (New England BioLabs, Catalogue No M0201S) , 10X NEBuffer 2.1 ( New England Biolabs, catalogue No B7202S) ,BsmB1 restriction enzyme (New England BioLabs, catalogue No R05800S), Aar1 restriction Enzyme (Fisher Thermo Scientific Catalogue no ER1581), 10X NEBuffer 3.1 (New England BioLabs , catalogue No B703S), (NEBR Stable Competent E. Coli (High Efficiency New England BioLabs) and Bovine Serum Albumin ( BSA) ,(New England BioLabs , catalogue No B9001S) was purchased from the NEW England BioLabs. The solution for cell culture was purchased from Gibco and it includes Dulbecco’s Modified Eagle Medium (Gibco, catalogue no, 41965-039), phosphate Buffered Saline (DPBS) solution (Gibco, catalogue no 31985-047) and Trypsin/ EDTA (T/E), (0.5%) (Gibco, catalogue no. 25300-054). T-75 Flask (Sarstedtr, catalogue No 83.3911.002) was bought from the Sarstedtr. DTT (Dithiothreitol), Cell Signaling Technology catalogue no 7016L, (4x Laemmli sample buffer, catalogue no 161-0747). Anti-Cas9 (7A9-3A3) mouse monoclonal Antibody (cell Signaling Technology, catalogue no 14697) was bought from Promeaga. Different type of antibodies were used Polyclonal goat Anti-mouse immunoglobulins HRP (Dako, catalogue no P044701-2), Anti-GAPDH rabbit polyclonal antibody (Sigma, catalogue no G9545) and LB broth (Fisher Scientific, catalogue no BP1426-500), HiSpeed Plasmid Midi Kit (Qiagen). Milk was prepared and purchased via the staff work in the super-lab. Agarose (Hi –Res Standard Agarose catalogue no AGD00010), 100x Tris/glycine premixed buffer, catalogue no 161-0734), SYBR (Safe DNA gel satin catalogue no 1771543). T- 25 (Sarstedtr catalogue no). Mini- Protein TGX precast Gels). Bio-Rad catalog 170-5060. T -75 were used with HEK293 media.
2-1 Amplification of Plasmids: The effective formulation of required recombinant plasmids necessitates an initial amplification phase of four different plasmid starting materials. Plasmids were obtained from Feng Zheng’s Laboratory and were subsequently subjected to culturing in a medium supportive of bacterial growth. Bacteria containing the required plasmids were isolated from the agar solid medium, and then cultured with 2 ml of LB broth and 100ml ampicillin in a 15 ml Falcon centrifuge tube. Subsequently, the bacterial suspensions were incubated in a shaking incubator at 37 ºC for 1 hour. 1 ml of the cultured LB broth was then removed from the Falcon tube and transferred to a 500-ml conical flask containing 50 ml of LB broth and 100 ml ampicillin. The flask was incubated in the shaking incubator at 37% for 12 hours in order to maximise bacterial growth. After 12 hours, 50 ml of LB broth now supporting an increased bacterial colony forming unit count were transferred to a 50 ml Falcon tube and centrifuged for 10 minutes at 3000 rpm. The supernatant LB broth medium was then discarded and the pelleted plasmid-containing bacteria retained. Speed Plasmid Midi Kit (Qiagen) was then utilised to extract pure plasmids from the cell pellet, with the concentration of final product being evaluated with a NanoDropm 8000 spectrophotometer (Thermo Scientific).
2-2 Digestion of Vectors The following components were mixed together to form the DNA vectors:
Reaction mix Volume µlPgRNAOligoAarIFast digest (FD) bufferH2OTotal
16 µl1.2 µl1 µl6 µl35.8 µl60 µl
Reaction mix Volume µlLenti guide –BsmBIFast digest (FD) bufferH2OTotal 10 µl1 µl6 µl43 µl60 µl The following mixtures were completed in duplicate form and were placed in a PCR machine at 37oC for 1hour. This was done to maintain the temperature at 37oC and not actually to undergo the PCR process.
An agarose gel was prepared at 0.8% with 50ml of TAE and two layers of loading slots. The top layer created was used to load the latter for pLenti-puro at the first and last slots, and the two duplications of the digested pLenti-puro and one undigested form used as a control were loading in the middle slots respectively. The bottom layer was used to load the latter for PgRNA modified in the same format as the top layer; at the beginning and the end. The two duplications of the digested form of the vector and one undigested sample of the vector used as a control were loaded in the middle slots. The gel was run for 90mins at 50V. After which, the intensity of the bands was measured.
2-2 Purification of Vectors: Care was taken to cut out the required DNA fragments from the digested vector carriers and place them in Falcon tubes. The agarose coating the DNA fragments was removed by melting in the presence of 4M potassium iodide during incubation for 10 minutes at 65°C on a dry block, with frequent mixing. After melting, the pH was neutralised by the addition of 350ul of buffer N3 (from the mini-prep kit). The resulting suspensions was then applied to a new mini-column. This tube was centrifuged at maximum speed for 1 minute, enabling the DNA fragments to bind to the membrane, after which, the supernatant was discarded. The membrane was subsequently rinsed with 700uL of buffer PB, containing ethanol. The suspension was then centrifuged once more and the supernatant again discarded. The membrane was then centrifuged for a third time to remove any residual ethanol. The contents of the mini-column were transposed to a new Eppendorf tube where 50ul of the elution buffer EB were applied for 1 minute prior to centrifuging for a further minute. The resulting eluent gave the digested, purified, required vector. The concentration of the resulting DNA was assessed as complete with the midi-preps. The PGRM 1/2, PLPG ½ annealed primers were diluted 1:200 by the addition of 2ul of the oligos and 400ul of distilled water.
2-3 DNA Ligation:
The ligation reaction was set up with the following components:pLenti-puro PgRNAPositive PGRM Positive PLPG Negative Control Positive PGRM Positive PLPG Negative Control50ng of DNA – 2ulAnnealed PGRM – 1ulQuick ligase buffer (X10) – 1ulLigase – 1ulddH2O – 5ulTotal 10ul 50ng of DNA – 2ulAnnealed PLPG – 1ulQuick ligase buffer (X10) – 1ulLigase – 1ulddH2O – 5ulTotal 10ul 50ng of DNA – 2ulQuick ligase buffer (X10) – 1ulLigase – 1ulddH2O – 6ulTotal 10ul 50ng of DNA – 3ulAnnealed PGRM – 1ulQuick ligase buffer (X10) – 1ulLigase – 1ulddH2O – 4ulTotal 10ul 50ng of DNA – 3ulAnnealed PLPG – 1ulQuick ligase buffer (X10) – 1ulLigase – 1ulddH2O – 4ulTotal 10ul 50ng of DNA – 3ulQuick ligase buffer (X10) – 1ulLigase – 1ulddH2O – 5ulTotal 10ul
The following mixtures were incubated at 4oC overnight.
2-4 Bacterial Transformation:The required recombinant plasmids were transformed to NEB® Turbo Competent (Escherichia coli) using the process recommended by the competent cells kit, which was guided by heat shock. After the recombinant plasmids were introduced into the competent cells, the transformed cells were plated out on LB agar plates with ampicillin and incubated at 37ºC for at least 12 hours. Following this incubation period, 10 individual colonies were selected from each agar plate and re-cultured in a 15 ml Falcon tube, with 2 ml of LB broth containing ampicillin. The transformation of the E. coli bacteria was carried so that the plasmids could be stored and replicated without any DNA rearrangement. The E. coli bacterial competent cells were then specifically treated to improve the transformation efficiency of the bacteria, rendering them more susceptible to transformation. The digested and purified pLentipuro, as well as the modified PgRNA, were added in duplicate to tubes containing the competent E. coli cells. Each tube was incubated in ice for 30 minutes and then subjected to heat shock treatment, where tubes were placed in a 42°C water bath for exactly 45 seconds, and then returned to the ice for 2 minutes. 950ul of SOC media were added to the tubes and the transformed cells were incubated at 37°C for a further 60 minutes with horizontal agitation at 200rpm. On completion of the incubation period, 150 ul of the bacterial mixture were plated out on to LB+AMP Agar plates and left incubating overnight at 30°C. This procedure caused only cells containing the plasmid to grow and form colonies on the agar plates. A single colony from each plate was selected and incubated separately in LB + Amp medium. A control plate was set up consisting of only the agar medium so that any difference in agar medium consistency following incubation could be demonstrated.
2-5 Cell Culture: Media Preparation: All bacterial lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplement with 1% L – Glutamine (200m M), 1% penicillin streptomycin (10,000U/ml) and 10 % Fetal Bovine Serum. A T -75 flask was used to set up the cell line and this will be incubated at 37 C with 5% CO2. Usually, the cell line was maintained by subculturing every three to four days. On completion of 15 passages, the cell line will be completely replaced from a new source.
2-6 Passaging cells:
HEK293 cell lines consisting of human embryonic kidney cells were grown at 370C with 8% CO2, in 20 ml of DMEM glucose rich media, contained in a 75ml flask. Cells were allowed to proliferate freely for two to four days, and following 15 passages, the media was renewed with a fresh 20ml aliquot to permit continued cell growth. 80-90 % confluence of cells was achieved after around 96 hours, and at this point the cell solution was divided in a 1/10 ratio. This was accomplished by initial removal of the media and subsequent rinsing of cells with 10 ml PBS. The PBS was then discarded and T/E (trypsin and EDTA) solution was pipetted directly onto the cells with gentle agitation to encourage their detachment from the bottom of the flask. 10 ml of media were added within 5 minutes of the T/E addition so as to neutralise its effect on the cells. This mixture was then transferred by pipette into a Falcon tube and centrifuged at 300 rpm for 5 minutes. The resulting supernatant was discarded and the remaining pellet tapped slightly to dislodge it. PBS was then added and the mixture was centrifuged once more to remove any residual media and trypsin from the cells. Again, the resulting supernatant was discarded, and 10 ml of new media added to the dislodged pellet. 1 ml of this sample was then diluted in 20 ml of media in a 75ml flask, producing a 1/10 dilution. Following separation of the cells, 10ul of the cell suspension and 10ul of Trypan Blue solution were combined in an Eppendorf tube. 10ul of this mixture were then transferred to a haemocytometer for colony counting, to assess cell proliferation. 2 ml of media in each well were estimated to contain around 300,000 cells. The cells were then plated out in triplicate and incubated once more at 370C with 8% CO2 for 48 hours.
2-7 Cell Transfection: These plates were then utilised in transfecting the cells with the constructed plasmids carrying the required sequences for the production of the guidance RNA, as well as the plasmid encoding the dCAS9-Dmnt3a sequence. Cell viability was observed to be greater than 90 % efficient for transfection. The DNA mixture containing Dcas9-Dnm3a (methylase), Dcas9-TET (demethylase) or PgRNA for each plate was diluted with reduced serum media, optimum, and combined with PEI (the transfecting reagent). This resulting complex generated was incubated for 30 minutes at room temperature. This complex mixture was then added to separate wells in the plate. DnM3a was applied to three wells, two wells were filled with TET and a further single well was filled with PgRNA. The plates were then gently agitated by shaking the plate back and forth. This procedure was repeated in triplicate. Finally, cells were incubated overnight at 37°C with CO2.Plate NO. Volume of Opti –MEM(µl) Volume of Dca9 –Dmnt3A, Dcas9- TET and pgRNA Volume of PEI1 600 µL 15 µL of Dcas9 methylase 45 µL2 400 µL 15 µL of Dcas9- TET 36 µL3 200 µL 5 µL of pgRNA 18 µL
2-8 Lactate Determination:The six well plates were used to generate media with different DNA in triplicate and subsequently incubated for 48 hours. Media samples were then transferred to Eppendorf tubes and any cell debris discarded following 1400 rpm centrifugation for 5 minutes. The supernatant was transferred to clean Eppendorf tubes. A lactate standard curve was generated using a 96 well plate by the addition of 0ul, 2.5ul, 5ul, 10ul, 20ul and 40ul diluted 1/10 with distilled water in triplicate. The triplicate samples were combined with 10ul in each well and further diluted 1/20. 100ul of lactate reagent were added to each well and left to incubate for 15 minutes at 370C. Finally, the plate was read at 540nm with the generated data being exported to an MS Excel worksheet for subsequent analysis.
2-9 Protein Extraction and estimation:
The following Six wells were generated within the plates to determine the protein concentrations that were generated by the Dcas9-TET and WI HEK293 cells. Following media removal, each well was washed with 1 ml of PBS for several minutes and then 100 ul of RIPA added, using the pipette tips to disrupt the cell structures to release the proteins contained. The solution from each well was then placed in Eppendorf tubes and any cell debris removed by centrifuging at 1400 rpm for 5 minutes. The supernatant was collected for protein determination: 10ul of each sample were placed in clean Eppendorf tubes and diluted with 50ul of distilled water. A BSA standard curve was generated in a 96 well plate by the addition of 0ul, 2.5ul, 5ul, 7.5ul, 10ul in triplicate. In addition, 10ul of each sample was added in triplicate. Around four to five sections of the 96 well plate contained the protein that was collected for each sample as follows: A – 5µl – WT HEK293B – 2 µl – WT HEK293C – 5 µl – dCAS9-TET HEK293D – 2 µl – dCAS9-TET HEK293
The BCA working reagent was generated by producing a 50:1 ratio of BCA with copper blue solution. 200 µl of the working BCA reagent was then placed in each well and incubated for 5 minutes at 550C. A plate reader, at 540nm, was used to assess the absorbance of each sample and again, the resulting data was exported to an Excel worksheet for subsequent analysis.
2-10 Gel Electrophoresis: A Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis was performed. This employed the samples that were previously examined during the protein determination, and utilised the previous calculations. These diluted samples were combined with an equal amount of distilled water, and then mixed further with an equal volume of 4x Laemmli buffer. The samples were then heated for 10 minutes at 95˚C in a heating block to generate a reaction within the solutions. To prepare the gel electrophoresis analysis, 10μL of the standard protein ladder were added to the first well. 20ul of each diluted sample were loaded beside the standard and run for 45 minutes at 170 V (need to double check). On completion of this stage, the transfer equipment kit was assembled. The gel was removed from the gel case and quickly washed in distilled water. A multi-layered filtration stage was prepared consisting of filter paper, then a nitrocellulose membrane and then the gel containing the samples, and finally a further filter paper. This was then saturated in the transfer buffer. The trans-blot turbo transfer system from Bio-Rad, in operation for 7 minutes, resulting in the transfer of the proteins on to the nitrocellulose membrane. The nitrocellulose membrane proteins were then washed with milk for 15 minutes to terminate the reaction. The nitrocellulose membrane was then rinsed three times with TTBS for 15 minutes. Primary antibody preparation was performed by dilution of the protein reagent antibody, MTCO2, by addition of 5 µl of the antibody to 10 ml of 10% milk. This mixture was applied liberally over the membrane followed by incubation overnight on a shaker at 40C. The membrane was then rinsed a further three times for 15 minutes each in TTBS. In addition, a secondary antibody was prepared by the addition of 1ul of anti-mouse catalogue A9044 in 10ml of 10% milk. This was applied over the membrane for 30 minutes. Once again, the membrane was rinsed three times with TTBS and prepared for Western Blot Analysis. This procedure was repeated utilsing the GAPDH housekeeping gene primary antibody on the membrane, and anti-rabbit secondary antibody.
2-11 Western Blot: Production of qualitative results from a Western Blot analysis requires that the amount of protein in each protein sample is identical, with the succession amount being around 20-50. Mixing of 1.5ml of Western ECL substrate peroxide reagent with ECL developer luminol/enhancer reagent, from Bio-Rad, in a Falcon tube produced a solution which was then pipetted onto the membrane. The ChemiDoc MP System was employed to generate the blot image for analysis, whilst the Image Lab TM software was used for processing (catalogue 170-8280).
Quantitative PCR
3- Results:
The sequence of the POLG gene is identified from the genome by browsing the human genome sequence, using the “gene browser” tool from genome.ucsc.edu. Figure 3 shows the genomic target of the CpG islands in the human POLG gene.
3-1 Titration curve of antibiotics:
To determine the suitable concentration of antibiotics for HEK293 cells, Excel is utilised to draw the titration curve of puromycin. The level of that killed roughly 90% of the normal of HEK293 cell would be chosen. Puromycin was able to do screen HEK293 cell that recombination with pgRNA.
Figure 3: Puromycin titration curve was used by Excel after 1-day treatment.
Bacterial Transformation:The following were showed a positively colonies which is means that bacterial were growthing in figure …
Figure 23: A photo bacteria colonies were growth
Figure4: The Fluorescence Microscopic images of transfection of HEK293 cell with dCAS9-Dmnt3a, Dcas9 TET and pgRNA plasmid at different ration
Fluorescence Imaging of Transfected cells:In order to determine the morphology after used Dcas9 – Damnt3a , dca9- TET and pgRNA cherry with DNA shown in figure 5 :
A B Figure 5: Panel assessment of DNA transfection on HEK293. The efficiency of HEK293 transfection was assessed by visual inspection of cell cultures expressing a red cherry fluorescence protein and panel B were Red fluorescence microphotograph of transfected HEK293 using the pgRNA modified plasmid.
3-3 Result of Wostren blot:Wostren blot brought to use in the quntitative analysis of the gene expression products which is protein, which can be used to identifiy the dcas9- Dmnt3A for pgRNA.
CtrI 1 2 Dcas9-Dmnt3A ( 118kDa)
CtrI Negative Control 1- Transfected cells before selection2- ………
GAPDH(37kDa) CtrI 1 2
Figure 6: the wostren blot results were transfected HEK293 with Dcas9-Dmnt3a so, all the band were from the same membrane and the density has been measured by the GelAnalyzer.
3-4 Restriction digest analysis: Restriciton enzymes BsmBI and Aar1 were empolyed and used in the double digestion of the pgRNA and Plenti- puro recombination plasmids. There were the first two was digested vector with BsmbI( 1.8kb + 8.2 kb) and the other was Ara1 (32 b + 8.3 Kb) and the following two was undigested plasmids:
MM #1 #2 Undigested Figure 7 : BsmbI digestion on pLentiPuroGuide (1.8 Kb + 8.2
MM #1 #2 Undigested Figure 8: AarI digestion on pgRNA (32 b + 8.3 Kb)
3-5 Assessment of methylation by q PCR:The following that If methylations occur, HpaII should not cut the CpG islands and PCR can be performed. If methylation is absent, then HpaII will cut the site and the PCR will be negative. Then, using flanking primers to the targeted sequences will be used in a Quantitative PCR using SYBR Green (Qiagen) as fluorescence detector (See figure 9 for details).
Figure 9 : Strategy for identification methylation changes in the POLG promoter region by q PCR
Figure 8:
Figure 11: this is specific gene which is dcas9 methylase with Polymerase gamma (polg).
Lactate determination:
Figure 20: in this curve were shown all the line was street that due to appear of fermentation
