Since the identification of the intermediate filament (IF), nestin, it has been used as a marker for Neural Stem Cells (NSCs) and commonly considered as a key component of the cytoskeleton. Custom animal models have enabled the discovery of nestin’s role in a multitude of biological functions, paving the way for potential therapeutic discovery. Herein, we review some publications that utilized custom mouse models to study the specific roles nestin plays across NSCs, the cytoskeleton, tendon differentiation, and tenogenesis.

How Nestin’s Role in NSC Survival Uncoupled from its Cytoskeletal Function

Researchers used Cyagen’s conditional knockout (cKO) mouse model to indicate the role in NSC survival is uncoupled from its structural involvement in the cytoskeleton through visualization of NSCs. Transgenic animals expressing green fluorescent protein (GFP) under the control of mouse Nes promoter (Nes-GFP transgene) were created to closely replicate endogenous Nes expression, serving as a marker for NSCs. Through additional breeding, researchers were able to generate Nes-GFP embryos with all possible genotypes for Nes. The in vivo function of nestin was elucidated through the generation of nestin-deficient mice, which causes embryonic lethality (compared to the overtly normal phenotypes of mice lacking type III or IV IF proteins – which are required for nestin to polymerize).  Embyronic lethality was confirmed to be due to increased apoptosis in NSCs with TUNEL assays and additional in vitro data.1

Given that nestin fails to polymerize into IFs in Vim-/- astrocytes, it was a surprising discovery that Vim-/- NSCs exhibited no difference in either cell expansion rate or caspase-3/7 enzymatic activity when compared with wild-type cells. Isolation of NSCs from Nes-/-;Vim-/- double knockout embryos were shown to display similar apoptotic phenotypes as Nes-/-, yet staining suggested that vimentin assembly/disassembly are not affected by nestin deficiency. This indicated that the role of nestin in the cytoskeleton – and its interactions with vimentin – are not required for nestin to function in the survival and self-renewal of NSCs. This study presented the finding that “microtubules, actin microfilaments, and vimentin network appear normal in Nes-/- NSCs.” This indicated that nestin deficiency had no detectable effect on cytoskeletal integrity - calling into question the previous emphasis on nestin’s role in cytoskeletal function.1

Implications of Nestin in Tendon Differentiation and Tendon Injury Repair

In another study, researchers were able to track the expression of nestin throughout postnatal development as well as under pathological conditions using a custom mouse model from Cyagen: homozygous transgenic mice (C57BL/6 strain) that express GFP under the control of the nestin promoter (Nes-GFP). In the Nes-GFP mouse Achilles tendon, “the number of Nes-GFP+ cells reached a peak on postnatal days 10 to 14… which decreased with age.” The pattern of nestin expression throughout development indicates nestin could play a role in tendon tissue differentiation. Additionally, nestin expression is shown to be activated at specific stages of tendon development, illustrating the potential role of nestin in future therapeutic strategies to treat tendon disease. To study nestin expression under pathologic conditions, the transgenic Nes-GFP mouse model of Achilles tendon injury was also used. GFP fluorescence revealed the accumulation of Nes-GFP+ cells at the injury site 1 week after injury, thereafter decreasing over time. Given the co-expression of tendon stem cell markers CD146 and CD105 in the Nes-GFP+ cells, the data suggests endogenous tendon stem cells may be activated upon injury. The collective findings of this study identified a subpopulation of nestin+ tendon stem/progenitor cells (TSPCs) that exhibited superior tenogenic capacity.2

Custom Mouse and Rat Models for Your Research

Whether your research involves the study of nestin - or additional compounds that have been implicated in a number of cellular or metabolic processes - the development of an appropriate rodent model can provide the investigative platform to help corroborate your next research breakthrough & identify new areas of potential therapeutics. Putative initiation and stop codons for the human nestin gene have been found at the same positions as in the rat gene, in regions where overall similarity was very high – such orthologous chromosomes provide valuable insights that are not reflected in the mouse genome. Additional studies of nestin in rat models could provide more elucidation into potential human therapeutics.

Cyagen offers a free targeting strategy design service to help you generate your customized rodent models (such as cKO, reporter knockin, point mutation, and/or humanized) to bring clarity to the role(s) your compound of interest plays in distinct cellular processes.

Please feel free to contact us to learn more about how we can help you generate quality mouse & rat models faster than any other approach available with a 100% guarantee of your specified genotype. With our proprietary TurboKnockout® mouse service, we can provide reliable large-fragment knockin (LFKI) up to 20 kb in a single round of gene targeting, providing F1 animals in as fast as 6 months.

 

References 

  1. Park, D., Xiang, A. P., Mao, F. F., Zhang, L., Di, C.-G., Liu, X.-M., … Lahn, B. T. (2010). Nestin Is Required for the Proper Self-Renewal of Neural Stem Cells. Stem Cells28(12), 2162–2171. doi: 10.1002/stem.541
  2. Yin, Z., Hu, J.-J., Yang, L., Zheng, Z.-F., An, C.-R., Wu, B.-B., … Ouyang, H.-W. (2016). Single-cell analysis reveals a nestin tendon stem/progenitor cell population with strong tenogenic potentiality. Science Advances2(11). doi: 10.1126/sciadv.1600874
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