<div><strong>Background:</strong> Ets variant factor 5 (ETV5) plays an important regulatory role in mouse Spermatogonial stem cells (SSCs) self-renewal. ETV5 knockout (KO) mice exhibit a progressive loss of SSCs and resulting in a Sertoli cell-only phenotype. The current study was aimed to use gene editing technology to obtain ETV5-KO pigs as a model for studying the apoptosis mechanism of SSCs and further clarify the function of ETV5 gene in pigs.<br />
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<strong>Methods:</strong> A gene editing plasmid for the porcine ETV5 gene was constructed, transfected into porcine fetal fibroblasts by electroporation to obtain ETV5-KO cells. ETV5-KO cells were used as donors to prepare ETV5-KO pigs by somatic cell nuclear transfer (SCNT). Testis tissues were collected for hematoxylin and eosin (HE), immunohistochemistry (IHC), RT-PCR testing and blood for ELISA testing from ETV5-KO pig.<br />
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<strong>Result:</strong> In the present study, we used the CRISPR/Cas9 system and SCNT to generate homozygous ETV5-KO pigs. We observed 3 phenotypes in these pigs: normal testis development after birth, the SSCs in the seminiferous tubules did not show obviously extinction at sexual maturity and normal spermatogenesis.</div>


<div>Generation of ETV5 Knockout Pigs with CRISPR/Cas9</div>


<p><span style="background-color:rgb(252, 252, 252); color:rgb(96, 96, 96); font-family:open sans; font-size:15px">Isolation of goat spermatogonial stem cells (gSSC) from pre-pubertal testes (3 to 6 months of age) by double enzymatic digestion is reported. The isolated cells were further enriched for spermatogonial stem cell population by filtration through 80- and 60-&micro;m nylon mesh filters, followed by differential plating on DSA-Lectin coated dishes. After overnight incubation, the unattached cells (putative gSSCs) were cultured on sertoli cell feeder layers in SSC medium, composed of DMEM supplemented with 10% FBS, GDNF (Glial cell line derived neurotrophic factor, 40 ng mL-1), bFGF (Basic fibroblast growth factor, 10 ng mL-1) and EGF (Epidermal growth factor, 10 ng mL-1). After 10-15 days, the putative SSCs started to develop and the cultures were maintained till the colonies stopped growing. The colonies were characterized for their affinity to Dolichos biflorus Agglutinin (DBA), alkaline phosphatase (AP) activity and various spermatogonial stem cell markers (PLZF, THY1, UCHL1, BCL6B and ID4). The developed colonies were positive for DBA, AP and all other markers, which is an indication of their being spermatogonial stem cells. In the present study, goat SSCs have been successfully isolated and characterized and the culture conditions will be improved further for developing more efficient and long term in vitro spermatogonial stem cell culture system in goat.</span></p>


<p>Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes</p>


<div>Livestock animal are important for agriculture economy and biomedical research. They are sources of Milk, meat, carcass, organic manure and other products. The development of genome editing technologies, especially CRISPR-Cas have revolutionized the generation of gene edited farm animals. In this review, we briefly introduce the CRISPR-Cas9 technology and highlight its application on livestock such as human disease modeling, disease resistant animal, and generation of hornless cattle, animal welfare and other agricultural and biomedical related traits which enhance the livestock production in order to meet the increasing demand of food worldwide. The ability to transfer sperm-producing stem cells or spermatogonial stem cells (SSCs) from a donor animal into the testes of a recipient male could have multiple applications. Production of BLG free milk in cattle provides a promising way to those who have allergy to cow milk. The knockdown of myostatin gene in different species like Sheep, Goat, Cattle and pig is very helpful in the economy of meat industry. Besides the several benefits of CRISPR-Cas9 technology, the risk factors and ethics issues related to this technology should be reconsidered before they enter into CRISPR era.</div>


Generation of ETV5 Knockout Pigs with CRISPR/Cas9

Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes

The Application of CRISPR/Cas9 Technology for Farm Animals: A Review