br Conclusion Cellular reprogramming is an inefficient proce

Conclusion Cellular reprogramming is an inefficient process and obtaining the rare reprogramming intermediates is a major hurdle in understanding this important event at the molecular level. The application of certain techniques that require higher cell numbers such as transcription factor URMC-099 immunoprecipitation (ChIP), DNase-sequencing or mass spectrometry approaches is therefore time and cost intensive. Here, we present an improved OKSM reprogrammable mouse model facilitated by the use of the rtTA3 which is characterized by a more than nine-fold higher reprogramming efficiency and significantly increased yields of the rare reprogramming intermediates without affecting reprogramming kinetics or the sequence of cell surface marker changes. Hence, we believe that the rtTA3-OKSM mouse model will become a useful tool for the isolation of the rare reprogramming intermediates at significantly higher yields and will consequently facilitate the study of the reprogramming process by enabling techniques that require a large number of cells.
Acknowledgements
Resource table
Resource details Blood samples were donated by a 55-year old female patient with clinically and genetically characterised Alzheimer\'s disease by the Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest (Hungary). The patient carries a heterozygous SNV in the PSEN1 gene, which leads to an amino-acid change (p.Val89Leu) in the protein sequence. The mutation has been shown to be associated with pathologically confirmed familial Alzheimer\'s disease, and computational modelling indicated that the mutation may alter the helical structure of PSEN1 protein (Queralt et al., 2002). The patient also carries two possible benign synonymous SNVs in Presenilin 2 gene (NM_000447.2(PSEN2):c.69T>C; rs ID: rs11405, homozygous and NM_000447.2(PSEN2):c.129C>T; rs ID: rs6759, heterozygous). To generate BIOT-7183-PSEN1 iPSC line (Fig. 1A) the four “Yamanaka reprogramming factors” OCT3/4, SOX2, KLF4, and C-MYC were delivered into PBMCs using the integration-free Sendai virus gene-delivery method (Yang et al., 2008; Fusaki et al., 2009). The iPSC-like colonies were picked after 20–27days post-transduction. Beginning from passage 5 of the iPSCs, the absence/presence of Sendai virus vector was analysed by RT-PCR using Sendai virus vector (SeV) - specific primers (Table 1). After 7 passages, the elimination of the reprogramming vector was confirmed in BIOT-7183-PSEN1 iPSC line which was selected for further analysis (Fig. 1B). The karyotype of the BIOT-7183-PSEN1 iPSC line was determined by Giemsa-banding, proving normal diploid 46, XX karyotype, without any detectable abnormalities (Fig. 1B). In parallel, the pathogenic NM_000021.3(PSEN1):c.265G>C mutation was confirmed with Sanger sequencing in the newly established iPSC line (Fig. 1C). Expression of pluripotency markers was examined by immunocytochemistry staining, using antibodies against human OCT3/4, E-CADHERIN, and NANOG (Fig. 1A). The in vitro spontaneous differentiation potential towards the three germ layer of the BIOT-7183-PSEN1 iPSC line was demonstrated by the expression of endodermal (GATA4), mesodermal (BRACHYURY) and ectodermal (βIII-TUBULIN) markers (Fig. 1A) (Itskovitz-Eldor et al., 2000; Carpenter et al., 2003).
Materials and methods
Acknowledgement We would like to thank for Dr. Mária Judit Molnár and Dr. Viktória Reményi (Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary) for collecting the patient blood samples and providing clinical data. This work was supported by grants from EU FP7 projects (STEMCAM, PIAP-GA-2009-251186; STEMMAD, PIAPP-GA-2012-324451; EpiHealth, HEALTH-2012-F2-278418; EpiHealthNet, PITN-GA-2012-317146); a national research project RG-IPI-2013_TP7/026, and Research Centre of Excellence - 11476-3/2016/FEKUT.
Resource table Resource details To generate the BIOT-4828-LOAD iPSC line (Fig. 1A) the four “Yamanaka reprogramming factors” OCT3/4, SOX2, KLF4, and C-MYC were delivered into PBMCs using the integration-free Sendai virus gene-delivery method (Yang et al., 2008; Fusaki et al., 2009). The iPSC-like colonies were picked after 20–27days post-transduction. Beginning from passage 5 of the iPSCs the absence/presence of Sendai virus vector was analysed by RT-PCR using Sendai virus vector (SeV) - specific primers (Table 1). After 7 passages, the elimination of the reprogramming vector was confirmed in BIOT-4828-LOAD iPSC line which was selected for further analysis (Fig. 1B).