Volume 4 • Issue 1 • 1000137 J Cell Sci Ther ISSN: 2157-7013 JCEST, an open access journal Open Access Research Article Cell Science & Therapy Koli.S, J Cell Sci Ther 2013, 4:1 http://dx.doi.org/10.4172/2157-7013.1000137 Keywords: c-Kit; Kit-GFP transgene construct; SSCs; Germ cell transplantation Introduction Spermatogenesis is highly complex, well-orchestrated and eicient biological process in which Spermatogonial Stem Cells (SSCs) undergo self-renewal, proliferation and diferentiation to produce virtually unlimited number of spermatozoa [1]. During this process, several genes (c-myc, c-Kit, Oct-4, Androgen Binding Protein (ABP), Cyclic- AMP Responsive Modulator (CREM), Protamine-1, Heat Shock Protein-70 (HSP-70), Phosphogylcerate Kinase-2 (PGK-2), Transition Nuclear Protein-2 (TNP-2) and Azoospermic Factor-1 (AZF-1) etc.) are known to be expressed in cell and stage speciic manner in the testis [1,2]. Among these genes, great attention has been focused in recent years on c-Kit, since it is indispensable right from the gonadal development to the diferentiation of SSCs. Lack of or poor expression of c-Kit is responsible for abnormal spermatogenesis and infertility in males. In certain cases, infertility has been attributed to functional defects in c-Kit [3, 4]. c-Kit SCF (Stem Cell Factor) signaling is essential for the survival of SSCs [3,4]. However, the molecular basis of male infertility arising from defects in c-Kit remains poorly understood. c-Kit, a type III Receptor tyrosine kinase (RTK) that has sequence and structural similarities to the platelet-derived growth factor receptor-β-polypeptide (PDGFRB) family of RTKs [5,6]. c-Kit is allelic to the W locus on the mouse chromosome-5 [7,8]. he 21-exon c-Kit gene encodes 5.15 kb transcript, which translates into a product of ~150 kDa protein with 979 amino acids [8-10]. In the testis of mouse, development, proliferation and migration of primordial germ cells (PGC’s) starts at 7.5 day post coitum (dpc) and c-Kit mRNA expression is detected in the PGCs, From 15 dpc to 3 day post-partum (dpp), c-Kit expression is markedly reduced which coincides with the period of gonocytes quiescence [3,8]. Synthesis of c-Kit mRNA and protein during testicular development is in concordant with the irst appearance of diferentiating SSCs (A 1 -A 4 ) which occur at 7 dpp. Studies on the transcription factors that enhances or represses c-Kit promoter activity would determine the cellular expression during normal and abnormal spermatogenesis, which will provide valuable clues about the mechanisms underlying the development and diferentiation of SSCs. Ater considering various transcription factors reported in the literature for optimal c-Kit activity, we designed germ cell speciic c-Kit transgene construct. For instance, the transcription of the c-Kit is controlled by regulatory sequences located in the 5’ lanking region of the gene [10,11]. his region includes both core promoter sequences and also tissue-speciic enhancer sequences. he Proximal Promoter Region (PPR) is located 58 bp upstream of translation start site in the mouse promoter region. he PPR contains consensus regulatory binding sites such as SP1, Ap-2 and short GA-rich elements [10-13]. *Corresponding author: Reddy KVR, Division of Molecular Immunology& Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), J.M.Street, Parel, Mumbai-400012, India, Tel: +91-22-24192016; Fax: +91-22-24139412; E-mail: reddyk@nirrh.res.in Received Febrauary 19, 2013; Accepted March 22, 2013; Published March 25, 2013 Citation: Koli S, Sikarwar AP, Babu MR, Reddy KVR (2013) Design and Molecular Characterization of C-Kit Transgene Construct during Spermatogenesis in Mice. J Cell Sci Ther 4: 137. doi:10.4172/2157-7013.1000137 Copyright: © 2013 Koli S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract In the postnatal testis, undifferentiated Type-A Spermatogonial Stem Cells (SSCs) differentiate into Type-B Spermatogonial Cells (SGCs) in the presence of c-Kit, which is expressed in a cell/stage speciic manner during spermatogenesis in mammals. c-Kit is subjected to tight transcriptional control as it is essential not only for germ cells but also for the development of hematopoietic stem cells and melanocytes. Although, c-Kit expression is a dynamically regulated process, the molecular basis for its transcriptional regulation during spermatogenesis remains mostly unidentiied. The aim of the study is to determine whether or not introduction of c-Kit expressing transgene construct into impaired c-Kit expressing SSCs isolated from W v /W v mutant mice will differentiate and colonize into testis of recipient mice. For this purpose, a 7.4 kb pG1-Kit-ORF-GFP transgene construct that contains a 4.4 kb c-Kit- ORF-GFP expression cassette was designed and its in vitro expression was conirmed by immunoluorescence and lowcytometry analysis. For comparison of the results, three control-GFP constructs, having 1.1 kb (lacking both c-Kit promoter and ORF), 4.1kb (lacking c-Kit promoter) and 1.4 kb (lacking c-kit-ORF) were also designed and introduced into SSCs by electroporation (EP). These SSCs were introduced into the testis of busulfan treated recipient infertile mice via rete using Germ Cell Transplantation (GCT) approach. It was observed that SSCs carrying c-Kit-ORF-GFP transgene insert were able to colonized and differentiated into other germ cell lineages in the seminiferous tubules of recipient mice as compared to controls. In conclusion, the testis of W v /W v mutant mice have the population of SSCs, wherein the function of c-Kit signaling is impaired. For the irst time we report that SSCs function in the recipient infertile mice could be resumed with the introduction of c-Kit-ORF-GFP transgene insert. Thus the present indings may have signiicant impact in understanding reproductive physiology and potential for novel future therapies in patients with testicular failure. Design and Molecular Characterization of C-Kit Transgene Construct during Spermatogenesis in Mice Swanand Koli 1 , Arun P Sikarwar 2 , Murali R Babu 1 and Reddy KVR 1 * 1 Division of Molecular Immunology & Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), J.M.Street, Parel, Mumbai-400012, India 2 Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Denmark