Cryobanking of primary somatic cells of elite farm animals - A pilot study in domesticated water buffalo (Bubalus bubalis)

Dua, Seema; Sharma, Priyanka; Saini, Monika; Rawat, Nidhi; Rajendran, Rasika; Bansal, Sonu; Wakil, Abubakar Muhammad; Beniwal, Madhuri; Parashar, Atul; Bajwa, Kamlesh Kumari; Selokar, Naresh L.; Kumar, Rajesh; Kumar, Dharmendra; Yadav, P. S.

doi:10.1016/j.cryobiol.2020.11.014

Cited by 10 publications

(3 citation statements)

References 18 publications

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“…The buffalo fibroblasts used in this study were isolated, cultured, characterised, and cryopreserved earlier by [ 27 ]. In brief, fibroblasts were isolated from skin-tissue biopsies taken from the underside of the tail, just above the anal region.…”

Section: Methodsmentioning

confidence: 99%

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

Punetha,

Kumar,

Saini

et al. 2023

Animals

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Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

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Section: Methodsmentioning

confidence: 99%

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

Punetha,

Kumar,

Saini

et al. 2023

Animals

Self Cite

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“…In other words, genetic drift is negatively correlated with population quantity (a tiny population number is characterized by very high genetic drift, and a large one by diminished genetic sampling error, fluctuating allele frequencies or Sewall Wright effect). A triangle of Sewall Wright effect, mono-or biallelic alteration and gene relocation serve as pivotal components of the mechanisms underlying intra-and inter-population transmissions of genes [14,15]. As a consequence, the N e parameters predominantly represent lower quantities of specimens than the animal censuses, which count the complete population numbers (N c ) in livestock species or breeds.…”

Section: The Biological Background For In Situ and Ex Situ Conservati...mentioning

confidence: 99%

Creating Ex Situ Protected Bioreservoirs as a Powerful Strategy for the Reproductive Biotechnology-Mediated Rescue of Threatened Polish Livestock Breeds

2023

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The current article presents the state of the art of the creation of bioreservoirs of cryopreserved somatic and stem cell lines and cryopreserved or lyophilized germplasm-based resources of selected farm animal species. It also presents the agricultural, biological and biotechnological determinants, and showcases the importance of the National Research Institute of Animal Production (NRIAP) in Poland in this process. The aforementioned bioreservoirs serve as an innovative research tool used for cryogenically or lyophilizogenically assisted and species-specific ex situ conservation. The latter ensures, in the long term, not only restitution, but also perpetuation of sustainable biodiversity that underlies genotypic and phenotypic, intra- and inter-population variability within pure-breeding herds of the national conserved livestock breeds.

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“…The choice of an appropriate cryoprotectant is essential for generating somatic resource biobanks that allow the development of studies for biodiversity conservation (Dua et al, 2021; Pereira et al, 2018). Therefore, the present study aimed to establish whether the reduction in concentration and the type of intracellular cryoprotectants associated or not with the presence of SUC alters the conservation of Puma somatic cells.…”

Section: Introductionmentioning

confidence: 99%

Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas

et al. 2022

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The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.

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Cryobanking of primary somatic cells of elite farm animals - A pilot study in domesticated water buffalo (Bubalus bubalis)

Cited by 10 publications

References 18 publications

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

Creating Ex Situ Protected Bioreservoirs as a Powerful Strategy for the Reproductive Biotechnology-Mediated Rescue of Threatened Polish Livestock Breeds

Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas

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