Teratoma Experiment
Purpose:
Pluripotent embryonic stem cells (ESC) are capable of differentiation not only to the three germ layers, but also to germ cells. If maintaining ESC in the proper culture condition, they can sustain their self-renewal capacity in vitro, and recapitulate the inner cell mass. Genetic manipulation has provided a vital tool for assessing the biological function of many genes at different stages during embryonic development.
Here, in our study we aim to evaluate the biological function of the Alkbh5 gene in maintaining the stem cell properties, so we used two clustered regularly interspaced short palindromic repeats (CRISPR) technology for the establishment of two clones where the Alkbh5 gene was completely depleted. These two clones exhibited phenotypes regarding cell growth and differentiation ability in vitro. Therefore, to assess our findings in vivo we aim to use teratoma formation experiments to confirm our in vitro results. Teratoma formation is hallmark and the best suited test to examine the pluripotency capacity of ESC. Teratoma is a benign tumor which includes cells from the three germ layers (ectoderm, mesoderm and endoderm). To date, it is considered the first step for assessing the pluripotency of ESC in vivo.
Distress:
During this experiment the animals will not experience substantial distress. Inoculation of cell lines will be conducted by sub cutaneous injections on the flank of the animal while under gas anesthesia. Due to the sub cutaneous nature of the allograft, the allograft is easily measureable and hence easy to monitor. Any signs of necrosis will be easily discerned. The animals will be sacrificed if they display signs of visible pain, -distress or decline in overall health due to illness.
Expected benefit:
Assessing the effect of the genetic manipulations described on ESC pluripotency will contribute to new knowledge on the biological function of newly described genes in maintaining stem cell properties. This will not only bring groundbreaking fundamental knowledge for research, but has potential future implications for stem cell therapies and can lead to new discoveries concerning genetic diseases.
Number of animals, and what kind:
We are aiming to use NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunodeficient mouse strain, and the total number with be 20 mice (5 in each group and the experiment will be repeated twice). Due to the origin of the ESC from a different strain (allograft), we are using NSG mice to assess teratoma formation.
3R:
Replacement; To understand how these mutations will affect the differentiation in vivo there is a need to let this mutated cell line to grow controlled in a living system (definition of stem cell pluripotency)
Reduction; we will inoculate the animals twice on the flank to reduce number of animals used in the experiment.
Refinement; the animal’s cages are equipped with environmental enrichment. We use Sevoflurane inhalation anesthesia instead of long lasting injected anestesia to avoid hypothermia. The animals will be euthanized and tumor taken out long before the tumor reaches a size regarded to be causing discomfort for the animals.
Pluripotent embryonic stem cells (ESC) are capable of differentiation not only to the three germ layers, but also to germ cells. If maintaining ESC in the proper culture condition, they can sustain their self-renewal capacity in vitro, and recapitulate the inner cell mass. Genetic manipulation has provided a vital tool for assessing the biological function of many genes at different stages during embryonic development.
Here, in our study we aim to evaluate the biological function of the Alkbh5 gene in maintaining the stem cell properties, so we used two clustered regularly interspaced short palindromic repeats (CRISPR) technology for the establishment of two clones where the Alkbh5 gene was completely depleted. These two clones exhibited phenotypes regarding cell growth and differentiation ability in vitro. Therefore, to assess our findings in vivo we aim to use teratoma formation experiments to confirm our in vitro results. Teratoma formation is hallmark and the best suited test to examine the pluripotency capacity of ESC. Teratoma is a benign tumor which includes cells from the three germ layers (ectoderm, mesoderm and endoderm). To date, it is considered the first step for assessing the pluripotency of ESC in vivo.
Distress:
During this experiment the animals will not experience substantial distress. Inoculation of cell lines will be conducted by sub cutaneous injections on the flank of the animal while under gas anesthesia. Due to the sub cutaneous nature of the allograft, the allograft is easily measureable and hence easy to monitor. Any signs of necrosis will be easily discerned. The animals will be sacrificed if they display signs of visible pain, -distress or decline in overall health due to illness.
Expected benefit:
Assessing the effect of the genetic manipulations described on ESC pluripotency will contribute to new knowledge on the biological function of newly described genes in maintaining stem cell properties. This will not only bring groundbreaking fundamental knowledge for research, but has potential future implications for stem cell therapies and can lead to new discoveries concerning genetic diseases.
Number of animals, and what kind:
We are aiming to use NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunodeficient mouse strain, and the total number with be 20 mice (5 in each group and the experiment will be repeated twice). Due to the origin of the ESC from a different strain (allograft), we are using NSG mice to assess teratoma formation.
3R:
Replacement; To understand how these mutations will affect the differentiation in vivo there is a need to let this mutated cell line to grow controlled in a living system (definition of stem cell pluripotency)
Reduction; we will inoculate the animals twice on the flank to reduce number of animals used in the experiment.
Refinement; the animal’s cages are equipped with environmental enrichment. We use Sevoflurane inhalation anesthesia instead of long lasting injected anestesia to avoid hypothermia. The animals will be euthanized and tumor taken out long before the tumor reaches a size regarded to be causing discomfort for the animals.