NOPR Collection:

CRISPR/Cas9 knock-in of GST-tagged human Noggin in the β-casein gene locus of bovine ear fibroblast cells

2018-11-12T09:48:24Z

Title: CRISPR/Cas9 knock-in of GST-tagged human Noggin in the β-casein gene locus of bovine ear fibroblast cells Authors: Park, Sung-Won; Do, Hyun-Jin; Choi, Wonbin; Kim, Hyun Jeong; Kang, Man-Jong; Seo, Han Geuk; Kim, Jae-Hwan Abstract: We developed knock-in vector system of human Noggin mature sequence with glutathione S-transferase (GST) containing factor Xa protease linker to facilitate the subsequent purification of recombinant protein. To achieve this, bovine ear fibroblast cells were isolated and transfection conditions were optimized by electroporation. To generate knock-in vector, human Noggin lacking its native signal peptide is fused to GST and foot and month disease virus 2A (F2A), and then inserted into bovine β-casein gene exon 3. We also generated enhanced green fluorescent protein (EGFP) expression vector of GST-human Noggin mature fused to β-casein signal peptide and F2A, and successfully detected recombinant human Noggin protein secreted into culture media, followed by factor Xa cleavage. Then, we co-transfected human Noggin knock-in vector with single-guided RNA and Cas9 expression vectors into bovine ear fibroblasts and obtained the stably-integrated colonies by antibiotic selection. PCR screening analysis revealed that 26 out of 35 colonies positively integrated human Noggin knock-in vector into bovine β-casein locus. One of positive clones was subjected to chromosome analysis, presenting normal karyotypes. Our data may provide the additional purification guideline of recombinant proteins by tagging GST with a protease linker sequence in the upstream of target genes and a high efficiency of integration ratio into bovine β-casein locus. Page(s): 553-560

Transcriptional activation of LGR5 gene by an engineered CRISPR-Cas9-based system induces hepatic-specific factors

2018-11-12T09:41:24Z

Title: Transcriptional activation of LGR5 gene by an engineered CRISPR-Cas9-based system induces hepatic-specific factors Authors: Kim, Dong-Ho; Kim, Kye-Seong; Ramakrishna, Suresh Abstract: Several new approaches for reprogramming or direct reprogramming somatic cells have been implemented during the last few years. Endogenous gene activation or repression can be achieved using dead clustered regularly interspaced short palindromic repeats associated protein 9 (dCas9) system fused with a transcriptional activating or repression domain. This study was undertaken to screen and validate efficient sgRNAs targeting reprogramming or direct reprogramming transcription factors by CRISPR-Cas9 based system. In this study, we designed and validated several individual single-guide RNA (sgRNA) targeting LGR5 and Yamanaka factors, such as Oct3/4, Sox2, c-Myc and Klf4, for effective transcriptional activation in mouse cells. Furthermore, we investigated the combination of effective sgRNAs for the synchronized effect of transcriptional activation on LGR5 gene and Yamanaka factors and achieved approximately 8.4-fold and 38-fold higher levels of mouse LGR5 and Oct3/4 upregulation, respectively, compared with the control. Further, we demonstrate that the activation LGR5 gene promoter induces known defined factors responsible for direct conversion of somatic cells to hepatocyte-like cells. We envision that our validation of effective sgRNAs will facilitate the development of mouse induced pluripotent stem cells (iPSCs) and novel findings of LGR5 as a potential candidate for direct conversion of somatic cells to hepatocyte-like cells. Page(s): 561-568

The anti-peptide relaxin antibodies for monitoring the well-being of the fetus in pregnant bitches

2018-11-12T09:38:49Z

Title: The anti-peptide relaxin antibodies for monitoring the well-being of the fetus in pregnant bitches Authors: Shruthi, N; Indhu, M; Shende, A M; Pawde, A M; Singh, Praveen; Bhure, S K Abstract: Pregnancy management is difficult in canines and there is a lack of methodology which would allow monitoring embryonic well-being. Relaxin (Rlx) is being reported for use in pregnancy diagnosis in different species. Here, we evaluated seven anti-Rlx peptide antibodies for detection of well-being of the fetus in bitches. Peptides were synthesized using solid phase peptide synthesis chemistry and the hyper-immune sera raised in chicken against peptides. In sandwich enzyme linked immunsorbent assay (ELISA), the chicken anti-Rlx P4 as a capture and rabbit anti-prorelaxin as detection antibody, gave better results in terms of differentiating the pregnant from non-pregnant bitches. Thirty five canine serum samples (21 non-pregnant, 11 pregnant and 3 males) were screened. Among the 11 pregnant, six delivered normally and the rest of the bitches aborted a few days after the serum collection with one or two dead fetuses. Among the 11 pregnant serum samples, 4 showed the absorbance above the cut-off value set for pregnancy, which delivered healthy puppies and five bitches showed the absorbance below the set cut-off value aborted after a few days of blood collection with one or two dead fetuses. The specificity of the assay was found to be 90.47% and the sensitivity of the assay for detecting the well-being of the fetus is 82%. The serum samples collected from those bitches having problems related to normal whelping, had shown absorbance below the set cut-off value. The results of the study indicate that relaxin is a useful marker, specifically for monitoring the pregnancy and tells about the well-being of the fetus. Page(s): 569-578

Enhanced soluble expression and effective purification of recombinant human interleukin-11 by SUMO fusion in Escherichia coli

2018-11-12T09:34:44Z

Title: Enhanced soluble expression and effective purification of recombinant human interleukin-11 by SUMO fusion in Escherichia coli Authors: Nguyen, Thi-Quy; Duong, Thu-Huong; Dang, Thi-Ngoc-Ha; Le, Ngoc-Giang; Le, Quynh-Giang; Do, Thi-Huyen; Nguyen, Van-Do; Le, Thi-Thu-Hong; Truong, Nam-Hai Abstract: Human interleukin-11 is a multifunctional cytokine applied for the clinical treatment of thrombocytopenia. However, IL-11 has been considered a difficult protein in to express in an Escherichia coli expression system. Here, we demonstrate a suitable construction for high production of recombinant human interleukin-11 (rhIL-11) in E. coli. An optimized codon gene encoding human IL-11 was inserted in-frame with the small ubiquitin like modifier (SUMO) protein in the pE-SUMO3 vector. The SUMO IL-11 fusion protein was entirely expressed in soluble form and reached 31.6% of total soluble protein in E. coli. The rhIL-11 protein with a purity of over 99% was obtained at high protein yields of 320 mg rhIL-11 per liter of bacterial culture. Bioactivity of rhIL-11, as determined by proliferation of a TF-1 cytokine-dependent cell line, was 4.17 x 10⁵ unit/mg, similar to the activity of the natural protein. Interestingly, the rhIL-11 was purified easily and effectively due to its selective precipitation from the reaction mixture. To the best of our knowledge, this is the first report demonstrating self-aggregating recombinant protein after cleavage from SUMO. Thus, expression of rhIL-11 fused with SUMO yielded greatly increased soluble production and convenient purification, and could offer a potential drug candidate for deployment in clinical trials. Page(s): 579-585