Background: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods: First, we modified the Tet-pLKO-Puro vector to make it easy("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.

... / The goal of the project was to establish knock down of mRNA in human mesenchymal stem cells. Since these cells are difficult to transfect, a viral approach is needed to achieve sufficient expression of e. g. shRNA in a high percentage of cells to allow for an efficient silencing of corresponding mRNAs. For this purpose for every gene product of interest, a number of shRNA clones have to be tested to detect an individual shRNA with sufficient efficacy. Lentiviral systems for shRNA approaches have recently become available. The principal advantage of the lentiviral system is that it allows gene silencing in nondividing cells and therefore expands the usefulness of the RNAi-based gene silencing system. Lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types. Since the viral transfection of MSCs is a time consuming process that involves transfection of 293 FT cells plus transduction of target cells, for this thesis the following approach was chosen: genes of interest were checked for expression in 293FT cells by RT-PCR. These gene products can be silenced in 293FT cells simply by transfection of shRNA clones and efficacy was subsequently tested by RT-PCR. Beyond this thesis then the project can proceed with effective clones to transduce primary MSCs with individual shRNA clones identified as effective silencing tool in this thesis.

Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is broadly agreed on, the efficacy of currently available tools limits the strength of conclusions drawn from these experiments. This thesis describes a method to optimize molecular tools for high-throughput screening, both for shRNA and sgRNA based reagents. Using large shRNA efficacy datasets, we first designed an algorithm predicting the potency of shRNAs based on sequence determinants. Combined with a novel shRNA backbone that further improves the processing of synthetic shRNAs, we built a library of potent shRNAs to reliably and efficiently knock-down any gene in the human and mouse genomes. We then went on to apply a similar approach to identify sgRNAs with increased activity. We complemented this with conservation and repair prediction to increase the likelihood of generating functional knock-outs. With these tools in hand, we constructed, sequence-verified and validated arrayed shRNA and sgRNA libraries targeting any protein coding gene in the human genome. These resources allow large-scale screens to be performed in a multiplexed or arrayed format in a variety of biological contexts. I have also applied these tools to identify therapeutic targets to circumvent cancer resistance to treatment in two different contexts. To overcome the shortfalls of single target therapy, I have developed multiplexed multidimensional shRNA screening strategy, where two genes are knocked down simultaneously in each cell. This strategy allows the identification of gene pairs that could be targeted in tandem to maximize therapeutic benefits. As a proof of concept, I have used it with a subset of druggable genes in melanoma cell lines. Moreover, we have applied our genome wide shRNA libraries to a different resistance context, stroma-mediated resistance to gemcitabine in PDAC. In this project, we performed screens in a PDAC-CAF coculture setting to try and identify cancer vulnerabilities specifically in the presence of stroma. Overall, the tools developed in this thesis allow for the efficient knockdown or knockout of any gene, both in an individual or combinatorial setting. Apart from providing a resource that will be useful for many fields, we have performed several proof-of-concept studies where we have applied our tools to identify potential cancer drug targets.

The hippocampus is an integral brain region for affective disorders. HCN1 protein shows age-dependent increase in expression resulting in an increase in I[subscript h] in the dorsal hippocampal CA1 region. TRIP8b knockout mice lacking functional HCN channels as well as both HCN1 and HCN2 knockout mice have been shown to display antidepressant-like behaviors. The mechanisms or brain regions involved in these alterations in behavior, however, are not clear. We developed a lentiviral shRNA system to examine whether knockdown of HCN1 protein, and therefore h-channels, in the dorsal hippocampal CA1 region is sufficient to produce antidepressant-like effects. We found that silencing of HCN1 gene resulted in physiological changes consistent with a reduction of I[subscript h] and increased cellular excitability of CA1 pyramidal neurons. Rats infused with lentiviral-shRNA-HCN1 in the dorsal hippocampal CA1 region displayed antidepressant- and anxiolytic-like behaviors. Using voltage-sensitive dye imaging, we found that knockdown of HCN1 in the dorsal hippocampal CA1 region led to enhancement of hippocampal activity in large regions of the dorsal hippocampus. Our results demonstrate that changed hippocampal network activity by local manipulation of HCN1 channels in dorsal hippocampus led to anxiolytic- and antidepressant-like behaviors and suggest that HCN1 channels could be a potential target for treatment of anxiety and depression. / text

The REarranged in Transfection (RET) tyrosine kinase is an important signalling protein for the development of neural crest-derived tissues such as the enteric and sympathetic nervous systems. RET is constitutively activated in multiple human tumour types, such as thyroid carcinomas and some non-small cell lung cancers. RET has 3 distinct isoforms, RET9, RET43 and RET51, which are named after the lengths of their unique C-terminal tails. Here, we investigate the role of RET in the TT thyroid carcinoma cell line, where it is a driver of tumourigenesis, and in the MiaPaCa-2 pancreatic carcinoma cell line, where RET is not driving tumour initiation, but may nonetheless have a profound effect on tumour progression. We generated lentiviral constructs for shRNAs that target either RET9 or RET51 specifically, or a common region shared by all RET isoforms. TT and MiaPaCa-2 cells were transduced using these lentiviral particles to create stable cell lines containing knockdowns of total RET, RET9, or RET51. Using a variety of morphological and biochemical assays, we found that RET expression is critical for TT cell survival, and that both RET9 and RET51 play significant roles in driving cell proliferation in TT cells. Conversely, RET is not critical for MiaPaCa-2 cell survival, and RET knockdown had no effect on MiaPaCa-2 proliferation. MiaPaCa-2 cells instead underwent dramatic morphological changes, from their normal spindle-like mesenchymal appearance to an increasingly flattened and epithelioid character, in response to RET9, RET51 or total-RET knockdown. The observed morphological changes were coupled with significantly reduced invasiveness through matrigel towards a source of chemoattractant, suggesting a critical role for RET in mediating cell invasiveness. These results suggest that RET may not only drive tumourigenesis, but can also enhance disease progression when expressed in other tumour types. We predict that RET may play critical roles in perineural invasion in pancreatic cancers, a process where cells invade along peripheral nerve fibers by following an increasing concentration of chemoattractants secreted by nerve and glial cells. Thus, RET may be a valuable target to slow, or stop this process, which would have significant clinical implications in a wide variety of cancers. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-08-30 11:45:41.969

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of EGFR is a common feature in variety of human cancers including lung, colorectal, breast, pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi) pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated through the transfection of three specifically designed short hairpin RNAs (shRNAs) against human EGFR. The shRNAs were specifically designed using bioinformatics tools and their individual knockdown efficacy determined through the introduction of an exogeneous based target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show substantial potential for the specific and efficient targeting of EGFR in human cancer cells.

Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or abrogated in vivo leukaemogenesis. Thus this work has identified novel roles for PI modulator genes in human AML with possible therapeutic potential.

The use of molecular biology as a means to advance agriculture has proven beneficial in many fields. However, the development lentiviral vectors that utilize a livestock promoter to express short hairpin RNA (shRNA) has been limited to date. The goal of this research project was to develop and characterize lentiviral bovine Pol III mir30 shRNA expression vectors for future use in livestock research. The bovine Pol III promoter (7sk, U6-2, or H1) was inserted directly upstream of a mir30 shRNA expression sequence in the lentiviral vector pNef-GT. A transient luciferase knockdown assay in human embryonic kidney (HEK) 293T cells was used to compare the functionality of these vectors. The bPol III mir30 shRNA expression vector was co-transfected with the pGL3 luciferase expression vector and the renilla expression vector pLB at a ratio of 5:10:1 respectively. The vectors were allowed 48 hrs to produce their respective products before luciferase activity was measured with the Stop-n-Glo Assay (Promega). Each bPol III promoter was able to express a functional shRNA resulting in a reduction of luciferase activity greater than 68 percent. The bH1 and bU6-2 Luc shRNA vectors were the most effective vectors when transfected with >76 percent (p-value <0.05) reduced luciferase activity. To confirm that these promoters were functional after integration into a bovine genome, recombinant lentivirus was made from these vectors. These particles were then used to transduce a bovine kidney (MDBK) cell line that expressed luciferase. After transduction, transgenic cells were selected by the addition of the antibiotic, Geneticin to the culture media until a population of 100 percent bPol III expression cells were observed for two passages and luciferase activity was measured. The 7sk promoter was the most effective bPol III promoter that reduced luciferase activity in these cells by 72 percent (p-value <0.05), while the bU6-2 and bH1 were moderately effective at reducing luciferase levels (37 percent, 46 percent respectively). These experiments were the first to quantify the bovine Pol III promoter function after integration into a bovine genome. While variability was observed, for livestock based research, the b7sk lentiviral vector appears to be the best choice to express a shRNA from the genome of a bovine genome.

The Myocyte Enhancer Factor-2, or MEF2, transcription factor family is necessary for the differentiation and regeneration of both skeletal and cardiac muscle tissue. The transcription factors in this family are responsible for the activation of many muscle specific growth factor-induced and differentiation genes. There are four individual isoforms of MEF2; MEF2A, -B, -C, and –D, and the roles of these individual transcription factors are not completely understood. Knockdowns of these individual isoforms revealed that a MEF2A knockdown mouse model displays severe myofibrillar defects in cardiac muscle. This knockdown also has shown that MEF2A is required for myogenesis in vitro, where the other 3 isoforms, -B, -C, and –D, are not necessary for this process. One method of knocking down MEF2A to study its roles further is through the use of short hairpin RNAs (shRNA). The purpose of my research was two-fold. First, in order to test the specificity of this shRNA method, an shRNA-resistant MEF2A over expression construct in an adenoviral vector was created to perform rescue experiments. Second, to compare individual MEF2 isoform knockouts to a complete knockout of the entire MEF2 family, a dominant negative construct was created in an adenoviral vector. In both cases, a pShuttle-CMV adenoviral vector was used. The results of this experiment can be used to further investigate the roles of MEF2A in both regeneration and differentiation of skeletal and cardiac muscle tissue.

Periodontitis potentially contributes to many systemic diseases. Specific gram-negative bacteria such as Porphyromonas gingivalis (P.g) and Treponema denticola (T.d) contribute to the initiation and progression of periodontal disease via factors such as NF-κB, TNF-α, IL-1β, and CXCL1. Down-regulation of these factors by natural compounds or short hairpin RNAs (shRNAs) reduces periodontal bacteria-induced inflammation. Our preliminary data indicated that P.gingivalis / lipopolysaccharide (LPS) stimulates chemokine CXCL1 production in macrophages. CXCL1 stimulates LPS-induced TNF-α expression, resulting in inflammation. We hypothesize that inhibiting the expression of CXCL1 will reduce LPS-induced TNF-α production. We recently demonstrated that a CXCL1 shRNA inhibits LPS-stimulated CXCL1 production in macrophages and leads to reduced expression of LPS-stimulated pro-inflammatory cytokines TNF-α and IL-1β. This indicates that CXCL1 shRNAs have potential as inhibitors of LPS-induced inflammation. Further studies are needed to confirm these as well as to identify the signaling pathway.