title: MicroRNA-mediated silencing of bovine NANOG and MBD3

authors: Wilson, Brooke Carol

subjects: microRNA

abstract: Embryonic stem cells (ESCs) are pluripotent due to their ability to differentiate into any cell type of the body, including functional gametes. These cells are also capable of infinite self-renewal and are highly receptive to genetic modification. The use of ESCs as a reproductive tool offers an exciting platform for farmers to efficiently amplify or introduce desirable agricultural traits within their herd. Unfortunately, all attempts to derive naive pluripotent ESCs from livestock animals have been unsuccessful. This is most likely attributed to a poor understanding of pluripotency specification in these animals. Nanog and Mbd3 are two proteins which are intimately involved in early embryonic development and pluripotency in the mouse. However, their specific function in livestock animals remains unknown. The primary aim of this research project was to determine the feasibility of using a microRNA-mediated silencing approach to knockdown these two proteins in a bovine model. 

Three candidate microRNAs, designed for each gene transcript, were cloned into the BLOCK-iT™ pcDNA™ 6.2-GW/EmGFP-miR expression plasmid. Before application in an in vivo system, each microRNA was transiently screened in vitro to determine its specificity and knockdown potential. For screening purposes, various attempts were made to generate a stable bovine NANOG expression fibroblast line using an inducible piggyBac transposon expression system. However, stable integration rates were lower than expected and all surviving and overexpressing cell clones eventually entered into a non-proliferate state of cellular senescence. The knockdown potential of the NANOG microRNAs was eventually determined by transient cotransfection with a constitutively active NANOG expression plasmid into a bovine embryonic fibroblast line. All three NANOG-specific microRNAs were able to efficiently knockdown NANOG protein expression. The highest knockdown efficiency was 89% as quantified by immunocytochemistry.  By contrast, a MBD3 knockdown effect was not visible after transient expression of the MBD3-specific microRNAs, likely due to the longevity of the MBD3 protein which has a half-life of around 48 hours.
 
The practicality of the BLOCK-iT™ microRNA expression system for functional gene analysis relies on its ability to generate stable knockdown cell lines which could then be used as donor cells for somatic cell nuclear transfer cloning and production of bovine knockdown embryos. Despite the slow proliferative nature of the stable transfectants and their frequent failure to expand during selection, we have been able to eventually generate GFP-microRNA expressing stable knockdown cell clones, suggesting that this system is still a feasible option for investigating the biological functions of NANOG and MBD3 in cattle.

text: MicroRNA-mediated silencing of bovine NANOG and MBD3 Embryonic stem cells (ESCs) are pluripotent due to their ability to differentiate into any cell type of the body, including functional gametes. These cells are also capable of infinite self-renewal and are highly receptive to genetic modification. The use of ESCs as a reproductive tool offers an exciting platform for farmers to efficiently amplify or introduce desirable agricultural traits within their herd. Unfortunately, all attempts to derive naive pluripotent ESCs from livestock animals have been unsuccessful. This is most likely attributed to a poor understanding of pluripotency specification in these animals. Nanog and Mbd3 are two proteins which are intimately involved in early embryonic development and pluripotency in the mouse. However, their specific function in livestock animals remains unknown. The primary aim of this research project was to determine the feasibility of using a microRNA-mediated silencing approach to knockdown these two proteins in a bovine model. 

Three candidate microRNAs, designed for each gene transcript, were cloned into the BLOCK-iT™ pcDNA™ 6.2-GW/EmGFP-miR expression plasmid. Before application in an in vivo system, each microRNA was transiently screened in vitro to determine its specificity and knockdown potential. For screening purposes, various attempts were made to generate a stable bovine NANOG expression fibroblast line using an inducible piggyBac transposon expression system. However, stable integration rates were lower than expected and all surviving and overexpressing cell clones eventually entered into a non-proliferate state of cellular senescence. The knockdown potential of the NANOG microRNAs was eventually determined by transient cotransfection with a constitutively active NANOG expression plasmid into a bovine embryonic fibroblast line. All three NANOG-specific microRNAs were able to efficiently knockdown NANOG protein expression. The highest knockdown efficiency was 89% as quantified by immunocytochemistry.  By contrast, a MBD3 knockdown effect was not visible after transient expression of the MBD3-specific microRNAs, likely due to the longevity of the MBD3 protein which has a half-life of around 48 hours.
 
The practicality of the BLOCK-iT™ microRNA expression system for functional gene analysis relies on its ability to generate stable knockdown cell lines which could then be used as donor cells for somatic cell nuclear transfer cloning and production of bovine knockdown embryos. Despite the slow proliferative nature of the stable transfectants and their frequent failure to expand during selection, we have been able to eventually generate GFP-microRNA expressing stable knockdown cell clones, suggesting that this system is still a feasible option for investigating the biological functions of NANOG and MBD3 in cattle.

year: 2015

title: The quantification of external colour changes during sexual transition in the protogynous spotty wrasse Notolabrus celidotus

authors: de Farias e Moraes, Carlos Eduardo

subjects: sex change

abstract: Hermaphroditism in fish has been well documented. Protogynous hermaphrodites (female to male sex change) are normally found in a polygamous social hierarchy. The male is the dominant within the group, followed by the largest female and it is common to observe aggressive behaviour as an exercise of dominance. Most of the studies relating to sex change used tropical species as models, however, little is known about temperate species. In this research, Notolabrus celidotus, a temperate protogynous wrasse, was used to investigate sex change. The overall goal was to develop an accurate methodology to assist with colour analysis of an external landmark. Ten males and ten females had pictures taken from the whole body. Digital photography and computational image analysis programs were used to help quantify the colour. Results were found through circular statistics, as hue is read as an angle. It was found that the anal fin presented the most significant mean variation of hue, within the body (female = 95.353 ± 3.16°; male = 141.138 ± 17.23°). The technique was then used to quantify the colour changes of the anal fin as individuals transitioned from female to male. Histological examination was also analysed and classified according to the transitional phase of every fish. The developed technique was able to track the colour changes and allocate them into distinct sexual transition stages. Statistical analysis showed that major colour changes began at the mid-transitional stage and progressed until the terminal phase male was achieved. The histological analysis of the gonads contributed to the study demonstrating internal changes through each sexual stage, additionally showing connection between the changes internally and externally. Through the development of a method to quantify external colour in relation to sexual state, this work creates opportunities for future studies. Investigations of sex change in this species can now be conducted without the need for invasive or destructive sampling to identify sex changing individuals. Further to this, individuals that are at different stages of sexual transition may be identified and selected for sampling by their external colour. Overall, this study was able to quantify and allocate external colour changes into distinct stages that map the restructure of the gonads during sex change.

text: The quantification of external colour changes during sexual transition in the protogynous spotty wrasse Notolabrus celidotus Hermaphroditism in fish has been well documented. Protogynous hermaphrodites (female to male sex change) are normally found in a polygamous social hierarchy. The male is the dominant within the group, followed by the largest female and it is common to observe aggressive behaviour as an exercise of dominance. Most of the studies relating to sex change used tropical species as models, however, little is known about temperate species. In this research, Notolabrus celidotus, a temperate protogynous wrasse, was used to investigate sex change. The overall goal was to develop an accurate methodology to assist with colour analysis of an external landmark. Ten males and ten females had pictures taken from the whole body. Digital photography and computational image analysis programs were used to help quantify the colour. Results were found through circular statistics, as hue is read as an angle. It was found that the anal fin presented the most significant mean variation of hue, within the body (female = 95.353 ± 3.16°; male = 141.138 ± 17.23°). The technique was then used to quantify the colour changes of the anal fin as individuals transitioned from female to male. Histological examination was also analysed and classified according to the transitional phase of every fish. The developed technique was able to track the colour changes and allocate them into distinct sexual transition stages. Statistical analysis showed that major colour changes began at the mid-transitional stage and progressed until the terminal phase male was achieved. The histological analysis of the gonads contributed to the study demonstrating internal changes through each sexual stage, additionally showing connection between the changes internally and externally. Through the development of a method to quantify external colour in relation to sexual state, this work creates opportunities for future studies. Investigations of sex change in this species can now be conducted without the need for invasive or destructive sampling to identify sex changing individuals. Further to this, individuals that are at different stages of sexual transition may be identified and selected for sampling by their external colour. Overall, this study was able to quantify and allocate external colour changes into distinct stages that map the restructure of the gonads during sex change.

year: 2019

title: He Ara Angitu: A Description of Literacy Achievement for Year 0 - 2 students in Total Immersion in Māori Programmes

authors: Rau, Cath R.

abstract: In response to the recommendations of the Literacy Taskforce Report (1999) and issues highlighted in the Green Paper - Assessment for Success in Primary Schools (1998), the Ministry of Education funded a project in 2000 and 2001 to develop a description of achievement in reading and writing for five-year-old Māori medium students.

This provided the opportunity to take a systematic comprehensive look at children’s literacy performance during the first two years of instruction and begin to identify reasonable expectations of progress in reading, written and oral language.

text: He Ara Angitu: A Description of Literacy Achievement for Year 0 - 2 students in Total Immersion in Māori Programmes In response to the recommendations of the Literacy Taskforce Report (1999) and issues highlighted in the Green Paper - Assessment for Success in Primary Schools (1998), the Ministry of Education funded a project in 2000 and 2001 to develop a description of achievement in reading and writing for five-year-old Māori medium students.

This provided the opportunity to take a systematic comprehensive look at children’s literacy performance during the first two years of instruction and begin to identify reasonable expectations of progress in reading, written and oral language.

year: 2001