TALEN‐mediated gene targeting in porcine spermatogonia

Tang, Lin; Bondareva, Alla; González, Raquel; Rodriguez‐Sosa, Jose R.; Carlson, Daniel F.; Webster, Dennis A.; Fahrenkrug, Scott C.; Dobrinski, Ina

doi:10.1002/mrd.22961

Cited by 19 publications

(28 citation statements)

References 85 publications

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“…Thus, while the treatments appeared safe, improvements are needed in methodology before HDR can be applied to DMD. In future studies, better results might be achieved with a higher dose and a different system of delivery such as the other CRISPR dog study in which 2x10 13 vg/kg was delivered intravenously [11], an earlier treatment window as mentioned above, a different type of vector delivery such as AAV [11,44,45], or improved techniques of HITI [46] or prime editing [47]. A combination of these improvements could lead to a higher percentage of HDR efficiency at both the DNA and protein levels.…”

Section: Discussionmentioning

confidence: 99%

“…Successful DMD gene editing also has been achieved using clustered regularly interspaced short palindromic repeats (CRISPR) coupled with an enzyme, typically caspase (Cas)-9, in cell culture, the DMD murine model (mdx) [5][6][7][8][9][10], and, most recently, in the deltaE50-MD dog model for DMD [11]. Transcription activator-like effector nucleases (TALEN) has also been tested in vitro [7,12,13].…”

Section: Introductionmentioning

confidence: 99%

“…Most of these gene-editing studies have relied on non-homologous end joining (NHEJ) to 'snip' out mutated and out-of-frame exons [11][12][13][14][15][16], with apparent highly efficient correction in the deltaE50-MD dog [11]. Others have used homology-directed repair (HDR), typically relying on a donor clone to integrate with the targeted region to correct the DMD gene mutation [6-8, 10, 17].…”

Section: Introductionmentioning

confidence: 99%

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Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

et al. 2020

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Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

et al. 2020

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“…Encouragingly, great improvements of technologies have been recently made on isolation, characterization, in vitro culture, and in vivo transplantation of SSCs will provide us a robust research platform to explore the function of ncRNAs in SSC self‐renewal (X. Li, Ao, et al, ; Mulder et al, ; Takashima & Shinohara, ; Zhang, Sun, & Zou, ). Remarkably, common genome editing techniques such as TALEN and CRISPR–Cas offer a rapid, efficient, and accurate genome engineering platform for functional characterization of putative ncRNAs in the context of established various models both in vitro and in vivo (Sato et al, ; Tang et al, ). We believe that more and more ncRNAs related to SSC self‐renewal will be discovered and functionally characterized, it will provide us a deeper insight into spermatogenesis and male infertility and pave a theoretical ground for applications in preservation and restoration of male fertility.…”

Section: Discussionmentioning

confidence: 99%

Noncoding RNAs: Potential players in the self‐renewal of mammalian spermatogonial stem cells

Bie

Wang

et al. 2018

Molecular Reproduction Devel

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Spermatogonial stem cells (SSCs), a unique population of male germ cells with self‐renewal ability, are the foundation for maintenance of spermatogenesis throughout the life of the male. Although many regulatory molecules essential for SSC self‐renewal have been identified, the fundamental mechanism underlying how SSCs acquire and maintain their self‐renewal activity remains largely to be elucidated. In recent years, many types of noncoding RNAs (ncRNAs) have been suggested to regulate the SSC self‐renewal through multiple ways, indicating ncRNAs play crucial roles in SSC self‐renewal. In this paper, we mainly focus on four types of ncRNAs including microRNA, long ncRNA, piwi‐interacting RNA, as well as circular RNAs, and reviewed their potential roles in SSC self‐renewal that discovered recently to help us gain a better understanding of molecular mechanisms by which ncRNAs perform their function in regulating SSC self‐renewal.

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“…The SSEA4‐positive cells were isolated from 90‐day‐old porcine testis tissues by flow cytometry with the antibody directed against SSEA4. As the previous report, 24 the germ cell gate was drawn around the distinctive germ cell population on the forward and side light scatter dot plot and cells within this gate were sorted. We got 30‐50% cells in the germ cell gate from the whole population.…”

Section: Methodsmentioning

confidence: 99%

Stage‐specific embryonic antigen 4 is a membrane marker for enrichment of porcine spermatogonial stem cells

Zhang

et al. 2020

Andrology

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Background Spermatogonial stem cells (SSCs), as tissue‐specific stem cells, are capable of both self‐renewal and differentiation and supporting the continual and robust spermatogenesis for male fertility. As a rare sub‐fraction of undifferentiated spermatogonia, SSCs share most molecular markers with the progenitor spermatogonia. Thus, the heterogeneity of the progenitor cells often obscures the characteristics of stem cells. Distinguishing SSCs from the progenitors is of paramount importance to understand the regulatory mechanisms governing their actions. Objectives The present study was designed to reveal that SSEA4 can be a marker for putative porcine SSCs that distinguished it from the progenitors and to build a sorting program for efficient enrichment of porcine SSCs. Methods To explore expression of SSEA4 within the undifferentiated spermatogonial population, we performed co‐immunofluorescent staining for SSEA4 and common molecular markers (VASA, DBA, PLZF, c‐KIT, and SOX9) in the 7‐, 90‐, and 150‐day‐old porcine testicular tissues. SSEA4‐positive cells were isolated from the 90‐day‐old porcine testes by flow cytometry. Immunofluorescent, RNA‐sequencing, and transplantation analysis were used to reveal that SSEA4‐positive fraction holds the stem cell capacity. Results We found that SSEA4 was expressed in a rare sub‐fraction of porcine undifferentiated spermatogonia, and RNA‐sequencing analysis revealed that the genes for stem cell maintenance and SSC‐specific markers (ID4 and PAX7) were up‐regulated in the SSEA4‐sorted fraction, compared with undifferentiated spermatogonia. In addition, germ cell transplantation assay demonstrated that SSEA4‐positive spermatogonia colonized in the recipient testicular tubules. Sorting of the undifferentiated spermatogonia with anti‐SSEA4 antibody resulted in a 2.5‐fold enrichment of SSCs compared with the germ cell gate group, and 21‐fold enrichment of SSCs compared with the SSEA4‐negative spermatogonia group. Conclusions Our findings revealed that SSEA4 is a new surface marker for porcine undifferentiated spermatogonia. This finding helps to elucidate the characteristics of porcine SSCs and facilitates the culture and manipulation of SSCs.

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TALEN‐mediated gene targeting in porcine spermatogonia

Cited by 19 publications

References 85 publications

Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

Noncoding RNAs: Potential players in the self‐renewal of mammalian spermatogonial stem cells

Stage‐specific embryonic antigen 4 is a membrane marker for enrichment of porcine spermatogonial stem cells

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