Rat Spermatogonial Stem Cell-Mediated Gene Transfer

Chapman, Karen; Saidley-Alsaadi, Dalia; Syvyk, Andrew; Shirley, James R.; Thompson, Lindsay M.; Hamra, F. Kent

doi:10.1007/978-3-662-45763-4_12

Cited by 6 publications

(21 citation statements)

References 20 publications

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“…Testis cell cultures were then used to isolate enriched populations of laminin-binding spermatogonia by established methods (Hamra et al 2008;Hamra et al 2002) that first deplete somatic testis cells from the germ cell population by negative selection on plastic and collagen, before positive selection for the spermatogonial stem cells based on their ability to bind to laminin (Hamra et al 2002). Laminin binding spermatogonia were then sub-cultured every 2 days for 3 passages on fresh gelatin-coated dishes prior to passaging onto irradiated DR4 MEFs (Chapman et al 2011).…”

Section: Isolating Undifferentiated Type a Spermatogoniamentioning

confidence: 99%

“…Long Evans rat spermatogonial lines were derived from freshly isolated laminin binding spermatogonia, as previously described in detail (Chapman et al 2011;Wu et al 2009). Here, a SG Medium formulation was used to sub-culture Long Evans rat spermatogonial stem cell lines: Dulbecco's Modified Eagle's Medium:Ham's F12 Nutrient Mixture (1:1), 6 ng/ml GDNF, 6 ng/ml FGF2, 100 μM 2-mercaptoethanol, 6 mM L-glutamine and a 1x concentration of the B27-Minus Vitamin-A Supplement solution (Chapman et al 2015).…”

Section: Spermatogonial Culturementioning

confidence: 99%

“…Here, a SG Medium formulation was used to sub-culture Long Evans rat spermatogonial stem cell lines: Dulbecco's Modified Eagle's Medium:Ham's F12 Nutrient Mixture (1:1), 6 ng/ml GDNF, 6 ng/ml FGF2, 100 μM 2-mercaptoethanol, 6 mM L-glutamine and a 1x concentration of the B27-Minus Vitamin-A Supplement solution (Chapman et al 2015). Spermatogonia were sub-cultured in SG Medium on feeder layers of DR4 MEFs (Chapman et al 2011;Wu et al 2009). Prior to transplantation, spermatogonia were harvested from MEFs and incubated for 2 hr on gelatin-coated plates in SG Medium to deplete feeder cells.…”

Section: Spermatogonial Culturementioning

confidence: 99%

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Long Evans rat spermatogonial lines are effective germline vectors for transgenic rat production

2017

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Long Evans rat strains are applied as research models in a broad spectrum of biomedical fields (>15,800 citations, NCBI PubMed). Here, we report an approach to genetically modify the Long Evans rat germline in donor spermatogonial stem cells. Long Evans rat spermatogonial lines were derived from freshly isolated laminin-binding spermatogonia. Laminin-binding spermatogonia were cultured over multiple passages on fibroblast feeder layers in serum-free culture medium containing GDNF and FGF2. Long Evans rat spermatogonial lines were genetically modified by transposon transduction to express a germline, tdTomato reporter gene. Donor rat spermatogonial lines robustly regenerated spermatogenesis after transplantation into testes of busulfan-treated, allogenic, Long Evans rats. Donor-derived spermatogenesis largely restored testis size in the chemically sterilized, recipient Long Evans rats. Recipient Long Evans rats stably transmitted the tdTomato germline marker to subsequent generations. Overall, Long Evans rat spermatogonial lines provided effective donor germline vectors for genetically modifying Long Evans rats.

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Section: Isolating Undifferentiated Type a Spermatogoniamentioning

confidence: 99%

Section: Spermatogonial Culturementioning

confidence: 99%

Section: Spermatogonial Culturementioning

confidence: 99%

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Long Evans rat spermatogonial lines are effective germline vectors for transgenic rat production

2017

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“…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).…”

Section: Introductionmentioning

confidence: 99%

“…Recent studies further demonstrate successful rederivation of rat strains from freeze dried sperm after storage at 4 o C for more than five years (Hirabayashi et al, 2005;Hochi et al, 2008;Kaneko and Serikawa, 2012). 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).…”

Section: Introductionmentioning

confidence: 99%

Optimization of Freezing Conditions for Cryopreservation of Rat Spermatogonial Stem Cell

Syvyk¹,

Djachenko²,

Syvyk³

2018

J microb biotech food sci

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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.

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Rattus norvegicus Spermatogenesis Colony-Forming Assays

Hamra

2016

Methods in Molecular Biology

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Rat Spermatogonial Stem Cell-Mediated Gene Transfer

Cited by 6 publications

References 20 publications

Long Evans rat spermatogonial lines are effective germline vectors for transgenic rat production

Long Evans rat spermatogonial lines are effective germline vectors for transgenic rat production

Optimization of Freezing Conditions for Cryopreservation of Rat Spermatogonial Stem Cell

Rattus norvegicus Spermatogenesis Colony-Forming Assays

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