The creation and validation of aptamers binding to murine 3T3-L1 Preadipocytes: preliminary implications for controlled cellular attachment, differentiation and cell fate
- Authors: Rubidge, Mark Lourens
- Date: 2017
- Subjects: Oligonucleotides , Fat cells , Stem cells , Ligand binding (Biochemistry) , Fluorimetry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65247 , vital:28714
- Description: The controlled seeding of a variety of stem cells in vitro has been reported to alter the patterns of their subsequent differentiation. This has been attributed to the control of the surface microenvironment onto which adherent stem cells are cultured, especially control of the proximal density of neighbouring cells. Simultaneously, advances in the generation of aptamers - synthetic ligand molecules developed using in vitro selection techniques targeting complex molecules - have aided in the production of molecules capable of selectively binding to a variety of commercial stem cell lines. Combining the aforementioned research fields, the project reported in this thesis aimed to generate DNA-based aptamers capable of assisting with the selective binding of murine 3T3- L1 preadipocytes to a solid surface. This was performed with a view to, eventually, control the seeding densities of the adherent preadipocytes on the surface of the tissue culture dish in subsequent researchers. In the process of meeting this goal, several optimisations of the in vitro process by which aptamers binding to cells are generated (Cell-SELEX) were performed: an analysis into a variety of methods used for the removal of the single stranded aptamer candidate sequences attached to the surface of 3T3-L1 preadipocytes, a comparison of methods for the generation of single-stranded aptamer sequences from double-stranded DNA template molecules and a method for quantifying the removed ssDNA from the cell surface. Their use is further reported in this work. Initially, it was determined that a fluorimetric evaluation of the unbound single stranded DNA was the optimum technique to use to evaluate the relative amounts of aptamer DNA binding to target cells during cell-SELEX; this arose from the release of DNA, and other cell lysate contaminates, which interfered UV/ Vis quantification. The evaluation into different methods of ssDNA removal from the cell surface showed that although trypsinisation of the cells demonstrated the highest level of aptamer detachment (quantified by fluorimetry), there is a decrease the number of potential targets that aptamers could attach to. The most common method for detaching bound DNA aptamer molecules from cellular targets reported in literature, the use of high temperatures, was selected for cell-SELEX to increase the variability in potential target sites on the cell surface. Using techniques optimised in this work, fluorescently-tagged single-stranded oligonucleotide aptamers were later generated with a positive selection pressure to bind to the surface of the 3T3-L1 preadipocytes, but not to their differentiated adipocyte counterparts. After eight cycles of cell-SELEX, fluorescent spectroscopic analysis depicted a 74 % binding retention of the selection pool in the positive preadipocyte selection pool, as opposed to a 0.69 % binding of sequences to the negative differentiated preadipocytes. Following the isolation and identification of candidate sequences, seven separate sequences were identified as being successfully generated from the selection process. Bioinformatic characterisation of these placed sequenced aptamer candidates into two separate families, that were then analysed in opposition to each for their binding affinity toward each other. Using fluorescently-tagged sequences, the binding selectivity of the generated aptamers was validated using both epifluorescent microscopy and confocal microscopy. At this stage, an aptamer sequence selected from prior in-house research to serve as a negative control also demonstrated significant binding to the extracellular matrix of both preadipocytes and mature adipocytes. 5’-thiolated aptamer sequences were used to form self-assembled monolayers on the electrode surfaces of the impedimetric Roche xCELLigence Real-Time Cell Analysis. The use of aptamer sequences to capture the seeded preadipocytes, demonstrated a slight increase in the extent of binding of the preadipocytes to the gold electrode surface and produced some preliminary indications of alterations to the pattern and rate of subsequent differentiation in the preadipocytes. This provides preliminary evidence that aptamers developed to bind specifically to a stem cell line in vitro show potential to be used as to capture said cell when cast in a self- assembled monolayer assembly. This provides a future opportunity to control the seeding densities of the cells in vitro. The effects of cellular differentiation at a set of predefined cellular densities can be demonstrated on a desired stem cell line. , Thesis (MSc) -- Faculty of Faculty of Science, Biotechnology Innovation Centre, 2017
- Full Text:
- Date Issued: 2017
- Authors: Rubidge, Mark Lourens
- Date: 2017
- Subjects: Oligonucleotides , Fat cells , Stem cells , Ligand binding (Biochemistry) , Fluorimetry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65247 , vital:28714
- Description: The controlled seeding of a variety of stem cells in vitro has been reported to alter the patterns of their subsequent differentiation. This has been attributed to the control of the surface microenvironment onto which adherent stem cells are cultured, especially control of the proximal density of neighbouring cells. Simultaneously, advances in the generation of aptamers - synthetic ligand molecules developed using in vitro selection techniques targeting complex molecules - have aided in the production of molecules capable of selectively binding to a variety of commercial stem cell lines. Combining the aforementioned research fields, the project reported in this thesis aimed to generate DNA-based aptamers capable of assisting with the selective binding of murine 3T3- L1 preadipocytes to a solid surface. This was performed with a view to, eventually, control the seeding densities of the adherent preadipocytes on the surface of the tissue culture dish in subsequent researchers. In the process of meeting this goal, several optimisations of the in vitro process by which aptamers binding to cells are generated (Cell-SELEX) were performed: an analysis into a variety of methods used for the removal of the single stranded aptamer candidate sequences attached to the surface of 3T3-L1 preadipocytes, a comparison of methods for the generation of single-stranded aptamer sequences from double-stranded DNA template molecules and a method for quantifying the removed ssDNA from the cell surface. Their use is further reported in this work. Initially, it was determined that a fluorimetric evaluation of the unbound single stranded DNA was the optimum technique to use to evaluate the relative amounts of aptamer DNA binding to target cells during cell-SELEX; this arose from the release of DNA, and other cell lysate contaminates, which interfered UV/ Vis quantification. The evaluation into different methods of ssDNA removal from the cell surface showed that although trypsinisation of the cells demonstrated the highest level of aptamer detachment (quantified by fluorimetry), there is a decrease the number of potential targets that aptamers could attach to. The most common method for detaching bound DNA aptamer molecules from cellular targets reported in literature, the use of high temperatures, was selected for cell-SELEX to increase the variability in potential target sites on the cell surface. Using techniques optimised in this work, fluorescently-tagged single-stranded oligonucleotide aptamers were later generated with a positive selection pressure to bind to the surface of the 3T3-L1 preadipocytes, but not to their differentiated adipocyte counterparts. After eight cycles of cell-SELEX, fluorescent spectroscopic analysis depicted a 74 % binding retention of the selection pool in the positive preadipocyte selection pool, as opposed to a 0.69 % binding of sequences to the negative differentiated preadipocytes. Following the isolation and identification of candidate sequences, seven separate sequences were identified as being successfully generated from the selection process. Bioinformatic characterisation of these placed sequenced aptamer candidates into two separate families, that were then analysed in opposition to each for their binding affinity toward each other. Using fluorescently-tagged sequences, the binding selectivity of the generated aptamers was validated using both epifluorescent microscopy and confocal microscopy. At this stage, an aptamer sequence selected from prior in-house research to serve as a negative control also demonstrated significant binding to the extracellular matrix of both preadipocytes and mature adipocytes. 5’-thiolated aptamer sequences were used to form self-assembled monolayers on the electrode surfaces of the impedimetric Roche xCELLigence Real-Time Cell Analysis. The use of aptamer sequences to capture the seeded preadipocytes, demonstrated a slight increase in the extent of binding of the preadipocytes to the gold electrode surface and produced some preliminary indications of alterations to the pattern and rate of subsequent differentiation in the preadipocytes. This provides preliminary evidence that aptamers developed to bind specifically to a stem cell line in vitro show potential to be used as to capture said cell when cast in a self- assembled monolayer assembly. This provides a future opportunity to control the seeding densities of the cells in vitro. The effects of cellular differentiation at a set of predefined cellular densities can be demonstrated on a desired stem cell line. , Thesis (MSc) -- Faculty of Faculty of Science, Biotechnology Innovation Centre, 2017
- Full Text:
- Date Issued: 2017
Genetic analysis and field application of a UV-tolerant strain of CrleGV for improved control of Thaumatotibia leucotreta
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
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