Derivation of conditionally immortalised mouse DRG cell lines.
Lead Research Organisation:
University of Sheffield
Department Name: Biomedical Science
Abstract
Rodents are used in academic and pharmaceutical research to obtain cells for culture and tissue samples for histology. Neuroscience research is particularly dependent on freshly isolated cultures of neurons prepared from. This is because neurons do not divide and thus cannot be continuously propagated in culture. Thus scientists continuously need to sacrifice rodent to provide neurons for their experiments. do not exist as cell lines. Pain is an important warning system to guard against tissue damage. Pathological pain, however, has no warning value and has huge economical, social and personal costs on society. !?Pain!? scientists study sensory neurons of the dorsal root ganglia (DRG) in order to discover new analgesic drug targets. Available DRG-derived cell lines do not represent a particular type of DRG neurons but rather provide a !?generic!? sensory neuron environment. They do not replace the need to sacrifice rodents for cultures to study other sensory neuron types.
The aim of this proposal is to generate immortalised neuronal cell lines from adult mouse DRG neurons. We aim to isolate enough clones to reflect the functional diversity of DRG neurons. We plan to use the established Immorto mouse which has been used by many groups to derive conditionally immortalised cell lines from various tissues. This genetically modified mouse strainis engineered to make a protein that forces all cells to divide including neurons. However, one drawback of the Immorto mouse is that all cells express the foreign protein and therefore a DRG culture from it will contain immortalised cells from neuronal and non-neuronal origins. we have devised a novel strategy in order to select against the survival and propagation of non-neuronal cell types from Immorto DRG cultures. The strategy is based on introducing the antibiotic resistance enzyme neomycin in DRG neurons only. In this way, only DRG neurons can survive growth conditions that include the antibiotic G418. The neomycin resistance enzyme will protect DRG neurons from by degrading G418. All other cells that do not contain the neomycin resistance protein will die leaving behind DRG neurons.
We believe that the impact of our proposal on reduction of animals use in research will be considerable and easy to realise. The generated DRG cell line can be deposited and be made available for academic researchers to validate and use in their research. We will also make them available for drug companies to replace acute cultures for screening and testing experiments.
The aim of this proposal is to generate immortalised neuronal cell lines from adult mouse DRG neurons. We aim to isolate enough clones to reflect the functional diversity of DRG neurons. We plan to use the established Immorto mouse which has been used by many groups to derive conditionally immortalised cell lines from various tissues. This genetically modified mouse strainis engineered to make a protein that forces all cells to divide including neurons. However, one drawback of the Immorto mouse is that all cells express the foreign protein and therefore a DRG culture from it will contain immortalised cells from neuronal and non-neuronal origins. we have devised a novel strategy in order to select against the survival and propagation of non-neuronal cell types from Immorto DRG cultures. The strategy is based on introducing the antibiotic resistance enzyme neomycin in DRG neurons only. In this way, only DRG neurons can survive growth conditions that include the antibiotic G418. The neomycin resistance enzyme will protect DRG neurons from by degrading G418. All other cells that do not contain the neomycin resistance protein will die leaving behind DRG neurons.
We believe that the impact of our proposal on reduction of animals use in research will be considerable and easy to realise. The generated DRG cell line can be deposited and be made available for academic researchers to validate and use in their research. We will also make them available for drug companies to replace acute cultures for screening and testing experiments.
Technical Summary
Rodents are used in academic and pharmaceutical research to obtain cells for culture and tissues for histology. Neuroscience research is particularly dependent on primary cultures of neurons prepared from rodents (mainly rats but increasingly from transgenic mice as well). This is because neurons do not divide and thus do not exist as cell lines. Pain is an important warning system to guard against tissue damage. Pathological pain, however, has no warning value and has huge economical, social and personal costs on society. !?Pain!? scientists study sensory neurons of the dorsal root ganglia (DRG) in order to delineate pain signalling and thus discover new analgesic drug targets. DRG neurons are a heterogeneous population of neurons with distinct functional and histochemical properties. However, published DRG-derived cell lines do not represent a particular type of DRG neurons but rather provide a !?generic!? sensory neuron environment. They do not represent all functional types of DRG neurons and thus do not replace the need for primary cultures to study other sensory neuron types.
The aim of this proposal is to generate conditionally immortalised neuronal cell lines from adult mouse DRG neurons. We aim to isolate and functionally characterise enough clones to reflect the functional diversity of DRG neurons. We plan to use the established H2Kb-ts-A58 Immorto mouse which has been used by many groups to derive conditionally immortalised cell lines from various tissues. However, one drawback of the Immorto mouse is that all cells express the Immorto transgene and therefore a DRG culture from it will contain immortalised cells from neuronal, glial and non-neuronal origins we have devised a novel strategy in order to select against the survival and propagation of non-neuronal cell types from Immorto DRG cultures. The strategy is based on expressing the bifunctional reporter gene ?Ogeo selectively in neuronal cells of the DRG using Cre recombinase. This will enable us to use antibiotic selection to purify clones of neuronal origin only.
We believe that the impact on the 3Rs will be considerable and easy to realise. The generated cell line can be deposited and be made available for academic researchers to validate and use in their research. We will also make them available for drug companies to replace acute cultures for in vitro screening and testing experiments.
The aim of this proposal is to generate conditionally immortalised neuronal cell lines from adult mouse DRG neurons. We aim to isolate and functionally characterise enough clones to reflect the functional diversity of DRG neurons. We plan to use the established H2Kb-ts-A58 Immorto mouse which has been used by many groups to derive conditionally immortalised cell lines from various tissues. However, one drawback of the Immorto mouse is that all cells express the Immorto transgene and therefore a DRG culture from it will contain immortalised cells from neuronal, glial and non-neuronal origins we have devised a novel strategy in order to select against the survival and propagation of non-neuronal cell types from Immorto DRG cultures. The strategy is based on expressing the bifunctional reporter gene ?Ogeo selectively in neuronal cells of the DRG using Cre recombinase. This will enable us to use antibiotic selection to purify clones of neuronal origin only.
We believe that the impact on the 3Rs will be considerable and easy to realise. The generated cell line can be deposited and be made available for academic researchers to validate and use in their research. We will also make them available for drug companies to replace acute cultures for in vitro screening and testing experiments.