Generation of transgenic cloned dogs using adipose-derived mesenchymal stem cells

Abstract

Since the birth of the first cloned dog Snuppy, valuable canids were produced by SCNT using adult somatic cells. In addition, genetically modified dogs were generated by SCNT using fetal fibroblasts. Fetal fibroblasts are preferred as nucleus donors for SCNT used in producing transgenic dogs because they have excellent proliferative ability, are capable of being genetically modified, and have the ability to produce live offspring. However, the donor transgenic cells become senescent and unusable because stable transgene-expression, homologous recombination or multiple transfections require a long time for in vitro culture. As an alternative to fetal cells, recent reports indicate that some mesenchymal stem cells (MSCs) lines can be maintained sufficiently long enough for homologous recombination events to take place. MSCs can proliferate for many passages in culture and show constant growth. Furthermore, MSCs have the ability to give rise several differentiated cell types. Thus the object of this study was to determine whether canine adipose–derived mesenchymal stem cells (cASCs) can be a suitable donor cell for producing transgenic cloned dogs. In several laboratory animals and humans, ASCs are of considerable interest because they are easy to harvest and can generate a huge number of cells from a small quantity of adipose tissue. ASCs have applications in various research areas, such as cell therapy and tissue engineering especially in bone reconstruction. In order to cASCs in SCNT, this study compared cellular proliferation rate, viability, cellular size and expression patterns of genes related to pluripotency and epigenetic modification between canine fetal fibroblasts (cFFs) and cASCs. The cFFs were established from fetuses of pregnant beagle at the 28th day. The cASCs were isolated from subcutaneous adipose tissue collected from the inguinal region of a healthy dog. The cASCs were characterized through flow cytometry to be positive for CD29, CD44, CD73, CD90 and CD105, but negative for CD31, CD34 and CD45. Proliferation pattern, cellular viability as well as cell size at each passage of cFF and cASC were compared when the culture reached confluence. In addition, real time-PCR was performed to investigate different mRNA transcripts expression in both cell lines. Moreover, the cASCs were evaluated as a potential donor cell using interspecies SCNT (iSCNT)

cASCs were cultured in two different culture media (RCMEP or DMEM) and used for iSCNT. Next, to generate transgenic cloned dog, cASCs were established from a transgenic cloned beagle produced by nuclear transfer of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cASCs expressed RFP gene and cell-surface marker characteristics of MSCs, including CD29, CD44 and thy1.1. Furthermore, the cASCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. Isolated cASCs were used for SCNT and after embryo transfer into recipient, RFP-expressing transgenic recloned beagle pups (Magic) were produced by nuclear transfer of cASCs derived from a transgenic cloned beagle (Ruppy1). Another purpose of this study is to determine the degree of genetic identity between the cloned (Ruppy1) and recloned (Magic) dogs and evaluated whether the RFP expression and CMV promoter methylation of these two transgenic dogs are age-dependent. To produce a transgenic dog that expresses neuron specific transgene, human synapsin 1 promoter as primarily neuron selective was chosen. Synapsin 1-RFP (SYN1-RFP) was introduced into cASCs via lentiviral vector infection. The SYN1-RFP cells were injected into enucleated in vivo matured dog oocytes and fused by electric stimulation. The fused-couplets were transferred into the uterine tube of five naturally estrus-synchronized surrogates. As results, the cFFs and cASCs differed in the number of generation but not in doubling times at all passages. The mean cell size of cASCs was significantly smaller than that of cFFs. Cellular viability and apoptosis were significantly lower in cASCs when compared to passage-matched cFFs. The level of HDAC1 transcript in cASCs was significantly higher than in cFFs, but expression of DNMT1 was not different between the two groups. OCT4 and SOX2 transcripts showed significantly higher expression in cASCs than in cFFs. Thus, canine adipose-derived stem cells (cASCs) are promising as donor cells for SCNT. With this in mind, cASCs were evaluated as a potential donor cell using interspecies SCNT (iSCNT). RCMEP cultured cells contained significantly higher amount of SOX2, NANOG, OCT4, DNMT1 and MeCP2 than DMEM cultured cells (P <0.05). However, there was no significant difference in the rate of development to blastocysts between the two groups. Thus, these results showed that altering gene expression levels in donor cells by changing the culture medium did not influence subsequent in vitro development of cloned embryos. In SCNT for generating transgenic cloned dog, one dog among five (20%) maintained pregnancy and subsequently gave birth of two healthy cloned pups. The present study demonstrated for the first time the successful production of transgenic cloned beagles by nuclear transfer of cASCs derived from a transgenic cloned beagle. Moreover, the degree of genetic identity between the cloned and recloned dogs showed that both cloned dogs carried a single copy and same integration site of the RFP gene. The transgene protein quantity of both transgenic dogs, showed no significant difference in the relative RFP expression between the 1-year-old Ruppy1 and the 1-year-old Magic. Also, transgene protein levels increased with aging of the two dogs, while promoter methylation status decreased with age. Gene expression and promoter methylation showed similar opposite profiles during growth of the two transgenic dogs. Lastly, neuron specific transgene-expressed dogs were generated by SCNT and three cloned pups (SYN1-RFP A, SYN1-RFP B, and SYN1-RFP C) were produced by natural delivery or C-sec. One of them is still alive, healthy and does not show any abnormalities. In this thesis, cASCs have superior proliferation patterns, epigenetic modification and pluripotency ability compared to cFFs and as well as capable of producing transgenic dogs by SCNT. Furthermore, cASCs can become a valuable resource to provide an unlimited supply of identical nuclei and to produce a neuronal degenerative disease model dog.