Objective To summarize the research progress of xenotransplantation.Methods Domestic and international publications about xenotransplantation were summarized and reviewed. Results Hyperacute xenograft rejection was a huge problem for xenotransplantation， but it could be alleviated if the organs or tissues of donor were genetically modified. So far the graft survival time differed greatly due to characteristics of different organ. Conclusions By reviewing the studies of relevant papers about xenotransplantation， a comprehensive understanding of research background and a suitable research direction of xenotransplantation can be supplied. The graft organs or tissues from genetically modified donors are expected to avoid or alleviate hyperacute xenograft rejection.
Objective To establish the heterologous recombinant embryonic stem cell （ES cell） with ghrelin receptor (GHS-R) gene deletion in order to study the function of the GHS-R gene. Methods PGK-neo cassette was replaced by TK-neo in X-pPNT agent. The target region was located in exon 1 and exon 2 of GHS-R. Two homologous arms were amplified from mouse ES cells genomic DNA and constructed into X-pPNT with SalⅠ/NotⅠand EcoRⅠ/BamHⅠ, respectively. ES cells were electrotransfected with the linearized targeting vector and screened with G418 and Gancyclovir. Finally, the positive ES cell clones were identified by PCR and sequencing. Results The X-pPNT-TK-neo vector was obtained. And two homologous arms were inserted correctly. Finally, 328 positive clones were obtained by G418 and Gancyclovir screening, and 3 clones were confirmed as GHS-R gene homologous recombination. Conclusion This study provides the necessary basis for the establishment of the GHS-R knock out mouse model and the further study on GHS-R gene function in vivo.
Objective To determine the effect of insulin-like growth factor-1 (IGF-1) on angiogenesis in mouse breast cancer model of lower and normal serum IGF-1 levels after using angiogenesis inhibitor ginsenoside Rg3 （GS Rg3）. Methods The breast cancer models were established in control mice and liver specific IGF-1 deficient (LID) mice by feeding DMBA and were treated with GS Rg3. Vascular endothelial growth factor (VEGF) and F8-RAg were detected by immunohistochemical method in breast cancer tissues. IGF-1 gene and angiogenesis relating genes were detected by gene chip in breast cancer and normal breast tissue. Results The incidence rate of breast cancer in LID mice was lower than that in control mice (P＜0.05). VEGF expression and microvessel density of LID mice were lower than those in control mice (P＜0.05). Compared to the control mice, IGF-1, FGF-1, TGF-β1 and HGF genes were increased, and FGFR-2, PDGF-A and PDGF-B genes were decreased in breast cancer of LID mice. After GS Rg3 treatment, VEGFa, EGF, EGFR, PDGF-A and FGFR-2 genes were increased, IGF-1 and TGF-β1 genes were decreased in breast cancer of LID mice compared with the control mice. Conclusion IGF-1 may be involved in mouse breast cancer progression and associated with the growth of blood vessels. Angiogenesis inhibitor may play an antitumor role by IGF-1 and TGF-β1.
AMP-activated protein kinase (AMPK) is involved in the development and progression of tumors including hepatocellular carcinoma (HCC). However, studies on AMPK and tumorigenesis were largely based on experiments in vitro or tumor xenografts model. Here, we introduce a liver-specific AMPKα1 knockout mice model, which is achieved by Alb-Cre recombinase system. The expression of AMPKα1 in the liver of AMPKα1-/--Alb-Cre mice is absent. AMPKα1 knockout in the liver does not affect the growth and histological structure of mouse liver. This model provides a favorable tool to the study of the roles of AMPKα1 in liver metabolism or tumorigenesis.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
Objective To construct an Escherichia coli outer membrane protein-A (OmpA) gene-deleted strain by Red homologous recombination, and laid the foundation for subsequent research. Methods Polymerase chain reaction (PCR) primers were designed according to the known OmpA gene sequence, and plasmid pKD3 for PCR amplification and integration; the fragment was transformed into Escherichia coli by λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation OmpA protein knocked out was observed by Western-blotting. Results The knock out gene product was correspond to a expected molecular weight. The western-blotting show that OmpA protein was knocked out. The difference in growth curve between the wild type and Escherichia coli △ OmpA gene-deleted strain was not significant. Conclusion OmpA gene deletion had no significant effect on the growth of Escherichia coli, which provides a foundation for further research on live vector vaccine.
The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.
ObjectiveTo explore the effect of transplanting neonatal porcine islet cells of pig via hepatic portal vein in type Ⅰ diabetic monkeys.MethodIn this study, three pig-monkey islet xenotransplantation experiments were carried out by using α-1, 3-galactosyltransferase (GGTA1) gene knockout neonatal pig islet cells.ResultsThree macaques were successfully transplanted with islet cells. After the operation, their vital signs were stable and no symptoms of venous embolism occurred. After transplantation, the blood glucose and the dosage of exogenous insulin were significantly reduced, and the specific porcine C-peptide could be detected. Three macaques developed symptoms of ketoacidosis, and one macaque developed wound infection. After symptomatic treatment, all of them survived for 16 weeks.ConclusionGGTA1 knockout neonatal porcine islet cells transplanted through hepatic portal vein is effective for the treatment of type Ⅰ diabetes.