Customized Transgenesis via Modification of Spermatogonial Stem Cells

Syvyk, Tetyana; Syvyk, Andrew

doi:10.15414/jmbfs.2018.7.5.475-479

Cited by 3 publications

(8 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Successful recovery of the mutant cell lines and ultimate reestablishment of the animal line are of paramount importance for any research project and for generation of mutant libraries especially [10,11]. The spermatogonial cell lines could be recovered from cryopreserved cultures and transplanted into an appropriate recipient for the reestablishment of mutant animal lines upon request [3,12]. For this purpose, we thawed several previously frozen collagen-none-binding primary spermatogenic cell cultures and applied a standard sequential differential adhesion selection on laminin and collagen-coated plates for initiation of the selected spermatogonial stem cell lines.…”

Section: Molecular and Cell Biotechnologiesmentioning

confidence: 99%

“…The study on spermatogenesis is advanced due to the ability to isolate, maintain, preserve, recover and transplant spermatogonial stem cells from different species [1][2][3]. There are three main methods developed for isolation of primary culture of spermatogonia from the variety of species based on different principals and their combinations: magnetic or fluorescent cell sorting based on surface markers [4]; density gradient centrifugation [5]; and differential adhesion selection [6].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Advancing recovery and cryopreservation of rat spermatogonia for germ stem cell banking

et al. 2018

View full text Add to dashboard Cite

Optimisation of spermatogonial cryopreservation medium and spermatogonia recovery procedures essential for the male germ cell research and spermatogonia mediated transgenesis in rodents. Methods. PCR, LIVE/DEAD® Cell Viability Assays, Spermatogonial Colony Forming Assay, immunocytochemistry, cryopreservation and thawing spermatogonia cells. Results. Three Sleeping Beauty mutant spermatogonia stem cell lines were stored in liquid nitrogen, after 17-24 months of cryopreservation, were derived in parallel from recovered cryopreserved cultures via an established laminin selection procedure and by an alternative method termed as direct SG (Spermatogonia Growth) medium selection on MEFs (mouse embrionic fibroblasts). A spermatogenic potential of the cell lines derived by these two methods was verified by the spermatogonia transplantation in vivo and reestablishing the mutant animal lines. We optimized the concentration of DMSO in a freezing medium for spermatogonial cell lines. Conclusions. We developed and optimized recovery of spermatogonial stem cells, that could be consistently derived from the freshly thawed stocks of testicular cells cryopreserved in SG medium. Our data suggest that the 8 % DMSO is the most effective concentration of the cryoprotectant in SG medium.

show abstract

Section: Molecular and Cell Biotechnologiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Advancing recovery and cryopreservation of rat spermatogonia for germ stem cell banking

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Historically, rats represent the most widely applied non-primate mammals to model disease processes (Chapman et al, 2011;Syvyk et al, 2017). Given the rate at which genetic know-how for editing rat genomes is accelerating, and the ever expanding rat genomics toolbox, strategies for preserving rat germline resources are becoming even more essential (Ivics et al, 2011;Izsvak et al, 2010;Syvyk et al, 2018). Freeze drying spermatozoa for rederiving rat strains appears especially attractive in that archiving samples do not require low temperature storage.…”

Section: Introductionmentioning

confidence: 99%

“…In addition to preserving sperm, unique biology of the testis allows it to mobilize mutant sperm production from large numbers of donor sperm stem cells, a trait being exploited to help build a genome wide bank of mutant rat germlines (Chapman et al, 2011;Stacey and Masters, 2008). Apart from studying spermatogenesis itself, male germ stem cell can be utilized for generation of random or predetermined genetic alterations and transfer those alterations onto a whole organism via direct germline transmission (Brinster and Nagano, 1998;Chapman et al, 2011;Wu et al, 2012;Syvyk et al, 2018). In this case, a robust and reproducible methodology for primary culture preparation and storage with subsequent line derivation is needed.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Freezing Conditions for Cryopreservation of Rat Spermatogonial Stem Cell

Syvyk¹,

Djachenko²,

Syvyk³

2018

J microb biotech food sci

Self Cite

View full text Add to dashboard Cite

Different cell types demonstrate an individual response to the doses of DMSO in freezing media that cannot be calculated empirically. Such situation requires experimental adjustment of the dose of the main cryoprotectant for each cell type. The purpose of the work was to optimize the basic composition of the cryopreservation medium for the rat spermatogonial cell lines, to investigate the time period of the initial storage of the cells at –80° C, as well as the effect of the ratio of volume of the freezing medium to the number of cells on their subsequent survival after recovery. Our data demonstrate that the 8% DMSO is the most effective concentration of the cryoprotectant. Furthermore, we tested different time of initial storage of cryovials in a commonly used “Mr. Frosty” Freezing Container at –80ºC. Our results suggest that 12 hours period (overnight) provides enough time for primary freezing step at –80ºC. Optimization of the volume of freezing medium revealed, that 0.5 ml, compared with 1.0 ml of medium for rat spermatogonial stem cells allows more effective preservation and long-time cryostorage of low numbers of cells (3×104) with successful recovery of up to 50% of the frozen cell population. Additionally, we attempted to improve the freezing medium composition by supplementing its base formulation with sucrose and/or trehalose. The use of optimal concentration of DMSO (8%) in the medium in combination with a 200mM of trehalose resulted in an increase of spermatogonia viability after recovery by more than 12% compared to the original composition of SG (Spermatogonia Growth) medium containing 10% of DMSO.

show abstract

“…High level of natural expression of ERBB2 in spermatogenic cells in an adult organism provides an opportunity to investigate additional aspects of ERBB2 facilitated pathways under habitual conditions. Beneficially, spermatogenic cell culture can serve not only as a model to investigate biological processes in vitro but can be utilized for spermatogonia mediated transgenesis that has a potential to generate genetically engineered organism in a wide variety of animal species for research in vivo [32]. Availability of more appropriate animal models can provide not only constant source of stable cell lines but bring the study on the level of the whole organism.…”

mentioning

confidence: 99%

Inducible dominant negative ErbB2 rat spermatogonial line for generation of transgenic rat model and dissecting ERBB2 tyrosine kinase mediated pathways

Syvyk

2023

Exp. oncol.

View full text Add to dashboard Cite

Summary. Aim: The ERBB2 receptors tyrosine kinase, also known as HER2/Neu, play an essential role in early organism development and modulation of cell behavior in varieties of tissue in an adult organism. Our aim was the generation of transgenic rat spermatogonial line capable of inducible expression of a dominant negative form of the ERBB2 protein in vitro and the transgenic rat as an animal model for dissection of the ERBB2 mediated signaling in vitro and in vivo. Materials and Methods: Donor derived rat spermatogonial stem cells that express green fluorescence protein and inducible ERT2CreERT2 recombinase were modified with Sleeping Beauty transposon-based vector that express truncated kinase deficient form of the ERBB2 receptor upon Cre mediated recombination. Clonally selected spermatogonial cell lines were extensively tested in vitro. Animals were generated via spermatogonia mediated transgenesis by transplantation of clonal cell line into testes of chemically sterilized recipients. Obtained progeny were tested for inducibility in vivo and served as donors of spermatogonia for downstream analysis. Results: We obtained animals and clonally derived spermatogonial stem cell lines that express an inducible dominant negative form of the ERBB2 protein. Isogenic nature of induced and uninduced cells allows most accurate morphological and molecular comparison of cells affected by the interruption of normal function of the ERBB2 receptor and cellular dynamics in vitro and in vivo. Conclusions: Clonally derived spermatogonial stem cell lines that express an inducible dominant negative form of ErbB2 demonstrated an obvious difference in morphological appearance and growth kinetics of induced cells comparing to uninduced ones. Western blot analysis of induced and uninduced cells revealed significant differences in presence and phosphorylation state of several tested important proteins involved in the ERBB2 mediated signal transduction, such as S6 ribosomal protein; AKT (the serine/threonine kinase also known as protein kinase B); and three protein kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade PRAS40, MEK, ERK1/2.

show abstract

Customized Transgenesis via Modification of Spermatogonial Stem Cells

Cited by 3 publications

References 7 publications

Advancing recovery and cryopreservation of rat spermatogonia for germ stem cell banking

Advancing recovery and cryopreservation of rat spermatogonia for germ stem cell banking

Optimization of Freezing Conditions for Cryopreservation of Rat Spermatogonial Stem Cell

Inducible dominant negative ErbB2 rat spermatogonial line for generation of transgenic rat model and dissecting ERBB2 tyrosine kinase mediated pathways

Contact Info

Product

Resources

About