Background: In the previous study, we established an ischemia-prone gerbil population(IG), which was selectively bred to increase the incidence of unilateral carotid arterial occlusion(UCO)-induced ischemia in Mongoli...Background: In the previous study, we established an ischemia-prone gerbil population(IG), which was selectively bred to increase the incidence of unilateral carotid arterial occlusion(UCO)-induced ischemia in Mongolian gerbils. However, if the characteristics of ischemia model in IG are the same as those in general gerbils(GG), and if the neurological symptoms are associated with the neurological insults in IG is still unclear.Methods: In the present study, we evaluated the UCO model in IG by analyzing neurological symptoms, neurological injury in the hippocampal CA1 region and compared with GG.Results: The data showed that the ratios of neurological symptom scores ≥ 2 in the IG and GG groups were 65.0% vs 30.0%, respectively, and were significantly different(P <.01).The neuronal damage following a UCO ischemic insult in the IG group was more severe compared to the GG group. There was a high correlation between the neurological insults' scale and the neurological symptom score in the IG and GG groups(r =.979 and.943 in the IG and GG groups, respectively). In animals with mild neurological symptom scores(2 and 3), the neuronal insults were significantly different between female and male gerbils in both IG and GG.Conclusion: Our findings suggest that IG population would likely be more advantageous to establish an ischemic model.展开更多
Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice ...Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice and 3 inbred strains of mice established in China was analyzed by polymerase chain reaction coupled with restriction fragment length polymorphism（PCR-RFLP） and PCR coupled with single-stranded conformation polymorphism（PCR-SSCP）. Results： With regard to the D-loop （Displacement loop, D-loop）, tRNA^ Met＋Glu＋Ile, and ND3 （NADH dehydrogenase subunit 3, ND3） gene fragments of mtDNA from these mice,no variation was revealed by PCR-RFLP at 46 restriction enzyme sites. Further analyzed by PCR-SSCP,the D-loop 5＇fragment and 3＇end fragment of mtDNA from these mice also showed no genetic variation. Conclusion： Owing to maternal mode of inheritance of mtDNA,the results indicate that these common inbred strains of mice share the same maternal lineage.展开更多
The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy.However,there are concerns about the safety of this system,specifically related to the use of guide-free Cas9.P...The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy.However,there are concerns about the safety of this system,specifically related to the use of guide-free Cas9.Previous studies have shown that guidefree Cas9 can induce genomic instability in vitro.However,the in vivo safety risks associated with guide-free Cas9 have not been evaluated,which is necessary for the development of gene therapy in clinical settings.In this study,we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo.Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo.The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein.Moreover,prolonged expression of Cas9 in pigs led to abnormal phenotypes,including a significant decrease in body weight,which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction.Furthermore,we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression,raising the risk of tumor occurrence.Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy.This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRiSPR/Cas9 system in clinical practice.展开更多
Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and no...Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and non-human primates proved unsuccessful.In the 1990s,scientists agreed that pigs were the most suitable donor animals for xenotransplantation.However,immune rejection between pig and human has hindered the application.To overcome these challenges,researchers developed genetically modified pigs that deactivate xenoreactive antigen genes and express human protective genes.These advances extended xenograft survival from days to years in non-human primates,resulting in the first human heart xenotransplant trial.Using genetically engineered pigs for the organ shortage is promising.This review provides an overview of potential incompatibilities of immunogenicity and functional proteins related to xenotransplantation between humans and pigs.Furthermore,it elucidates possible approaches for multiplex gene modification to breed better-humanized pigs for clinical xenotransplantation.展开更多
Background It remains almost a helpless situation for the recurrent implantation failure and pregnancy loss caused by endometrial injury at present. The purpose of this study was to develop a rabbit model of endometri...Background It remains almost a helpless situation for the recurrent implantation failure and pregnancy loss caused by endometrial injury at present. The purpose of this study was to develop a rabbit model of endometrial mechanical injury that could provide a research platform for this difficult clinical predicament. Methods Three experiments were conducted. Experiment 1： Curettages in both uterus horns and copper wire inserting after curettage （double-injury） in one horn. The histological changes were monitored at 0, 24, 48, 72 hours, as well as in 1 and 2 weeks after operation. Experiment 2： Direct copper wire inserting in one horn and double-injury in other horn. The wires in both horns were removed after 2 weeks.展开更多
The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interestingcandidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic...The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interestingcandidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cellnuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs expressapproximately 15–25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was alsomarkedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs withinfluenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profounddecrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-foldreduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found thatthe Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These resultsdemonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate thatthe Mx1 transgene can be harnessed to develop disease-resistant pigs.展开更多
Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous...Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.展开更多
Objective: To observe effects of Dan Wei Powder (胆胃散 Powder for treating the gall bladder and stomach) Tea Bag (DWSTB) on the aggregation rate of blood platelet in vivo and in vitro. Methods: Increase of the platel...Objective: To observe effects of Dan Wei Powder (胆胃散 Powder for treating the gall bladder and stomach) Tea Bag (DWSTB) on the aggregation rate of blood platelet in vivo and in vitro. Methods: Increase of the platelet aggregation rate in the rat in vivo was induced by carrageenin, and increase of the rabbit platelet aggregation rate in vitro was induced by adenosine diphosphate (ADP) and collagen, respectively. The effects of DWSTB on the platelet aggregation rate were investigated in vivo and in vitro, respectively. Results: The maximum in vivo platelet aggregation rate in the rat was significantly decreased after administration of 2.0 and 4.0 g·kg-1 DWSTB (P<0.05.P<0.01). The maximum rabbit platelet aggregation rate induced by ADP and collagen in vitro were suppressed significantly by 2.0-16.0 mg·mL-1 and 2.0-8.0 mg·mL-1 DWSTB, respectively (P<0.05.P<0.01). And the effect of DWSTB on platelet aggregation was raised with increase ofits dose. Conclusion: Dan Wei Powder Tea Bag can restrain the aggregation of platelet in vivo and in vitro.展开更多
基金Key Projects in the National Science&Technology Pillar Program,Grant/Award Number:2015BAI09B01
文摘Background: In the previous study, we established an ischemia-prone gerbil population(IG), which was selectively bred to increase the incidence of unilateral carotid arterial occlusion(UCO)-induced ischemia in Mongolian gerbils. However, if the characteristics of ischemia model in IG are the same as those in general gerbils(GG), and if the neurological symptoms are associated with the neurological insults in IG is still unclear.Methods: In the present study, we evaluated the UCO model in IG by analyzing neurological symptoms, neurological injury in the hippocampal CA1 region and compared with GG.Results: The data showed that the ratios of neurological symptom scores ≥ 2 in the IG and GG groups were 65.0% vs 30.0%, respectively, and were significantly different(P <.01).The neuronal damage following a UCO ischemic insult in the IG group was more severe compared to the GG group. There was a high correlation between the neurological insults' scale and the neurological symptom score in the IG and GG groups(r =.979 and.943 in the IG and GG groups, respectively). In animals with mild neurological symptom scores(2 and 3), the neuronal insults were significantly different between female and male gerbils in both IG and GG.Conclusion: Our findings suggest that IG population would likely be more advantageous to establish an ischemic model.
基金Supported by Developmental Programming Item of National Keystone Basic Research (G2000016106) and National Natural Science Foundation of China (No. 39600079)
文摘Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice and 3 inbred strains of mice established in China was analyzed by polymerase chain reaction coupled with restriction fragment length polymorphism（PCR-RFLP） and PCR coupled with single-stranded conformation polymorphism（PCR-SSCP）. Results： With regard to the D-loop （Displacement loop, D-loop）, tRNA^ Met＋Glu＋Ile, and ND3 （NADH dehydrogenase subunit 3, ND3） gene fragments of mtDNA from these mice,no variation was revealed by PCR-RFLP at 46 restriction enzyme sites. Further analyzed by PCR-SSCP,the D-loop 5＇fragment and 3＇end fragment of mtDNA from these mice also showed no genetic variation. Conclusion： Owing to maternal mode of inheritance of mtDNA,the results indicate that these common inbred strains of mice share the same maternal lineage.
基金This work was financially supported by National Key Research and Development Program of China(2022YFA1105403,2022YFA1105402,2021YFA0805903,2023YFF0724703,2021YFF0702601)Research Unit of Generation of Large Animal Disease Models,Chinese Academy of Medical Sciences(2019-12M-5-025)+4 种基金National Natural Science Foundation of China(32170542,32300426)Major Science and Technology Projects of Hainan Province(ZDKJ2021030)Science and Technology Planning ProjectofGuangdong ProvinceC,hina(2023B1212060050,2021B1212040016,2021A1515110909)Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City(2021JJLH0085,2021JJLH0096)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(Y2023096).
文摘The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy.However,there are concerns about the safety of this system,specifically related to the use of guide-free Cas9.Previous studies have shown that guidefree Cas9 can induce genomic instability in vitro.However,the in vivo safety risks associated with guide-free Cas9 have not been evaluated,which is necessary for the development of gene therapy in clinical settings.In this study,we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo.Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo.The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein.Moreover,prolonged expression of Cas9 in pigs led to abnormal phenotypes,including a significant decrease in body weight,which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction.Furthermore,we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression,raising the risk of tumor occurrence.Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy.This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRiSPR/Cas9 system in clinical practice.
基金supported by the National Key Research and Development Program of China(2022YFA1105404,2021YFF0702601 and 2021YFA0805300)the Research Unit of Generation of Large Animal Disease Models,Chinese Academy of Medical Sciences(2019I2M-5-025)+3 种基金the Science and Technology Program of Guangzhou(202201010409)the Key Research&Development Program of Hainan Province(ZDYF2021SHFZ052)the Major Science and Technology Project of Hainan Province(ZDKJ2021030)the 2020 Research Program of Sanya Yazhou Bay Science and Technology City(202002011)
文摘Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and non-human primates proved unsuccessful.In the 1990s,scientists agreed that pigs were the most suitable donor animals for xenotransplantation.However,immune rejection between pig and human has hindered the application.To overcome these challenges,researchers developed genetically modified pigs that deactivate xenoreactive antigen genes and express human protective genes.These advances extended xenograft survival from days to years in non-human primates,resulting in the first human heart xenotransplant trial.Using genetically engineered pigs for the organ shortage is promising.This review provides an overview of potential incompatibilities of immunogenicity and functional proteins related to xenotransplantation between humans and pigs.Furthermore,it elucidates possible approaches for multiplex gene modification to breed better-humanized pigs for clinical xenotransplantation.
文摘Background It remains almost a helpless situation for the recurrent implantation failure and pregnancy loss caused by endometrial injury at present. The purpose of this study was to develop a rabbit model of endometrial mechanical injury that could provide a research platform for this difficult clinical predicament. Methods Three experiments were conducted. Experiment 1： Curettages in both uterus horns and copper wire inserting after curettage （double-injury） in one horn. The histological changes were monitored at 0, 24, 48, 72 hours, as well as in 1 and 2 weeks after operation. Experiment 2： Direct copper wire inserting in one horn and double-injury in other horn. The wires in both horns were removed after 2 weeks.
基金This work was supported by grants from National Basic Research Program of China(973 program)(2011CB944203)ZNGI-2011-010 from the Guangzhou Municipality and the Chinese Academy and Sciences to L.L.
文摘The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interestingcandidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cellnuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs expressapproximately 15–25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was alsomarkedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs withinfluenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profounddecrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-foldreduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found thatthe Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These resultsdemonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate thatthe Mx1 transgene can be harnessed to develop disease-resistant pigs.
基金the National Key Research and Development Program of China(2017YFA0105103,2021YFA0805903)the National Natural Science Foundation of China(81941004,32170542)+10 种基金2020 Research Program of Sanya Yazhou Bay Science and Technology City(202002011)Major Science and Technology Projects of Hainan Province(ZDKJ2021030)Key Research&Development Program of Hainan Province(ZDYF2021SHFZ052)Youth Innovation Promotion Association of the Chinese Academy of Sciences(2019347)Young Elite Scientist Sponsorship Program by CAST(YESS20200024)Biological Resources Progaramme,Chinese Academy of Sciences(KFJBRP-017-57)Key Research&Development Program of Bioland Laboratory(Guangzhou Regenerative Medicine and Health Guangdong Laboratory)(2018GZR110104004)China Postdoctoral Science Foundation(2020M682943)Science and Technology Planning Project of Guangdong Province,China(2019A030317010,2020B1212060052,2021B1212040016,2021A1515011110)Science and Technology Program of Guangzhou,China(202007030003)Research Unit of Generation of Large Animal Disease Models,Chinese Academy of Medical Sciences(2019-I2M-5-025)。
文摘Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.
文摘Objective: To observe effects of Dan Wei Powder (胆胃散 Powder for treating the gall bladder and stomach) Tea Bag (DWSTB) on the aggregation rate of blood platelet in vivo and in vitro. Methods: Increase of the platelet aggregation rate in the rat in vivo was induced by carrageenin, and increase of the rabbit platelet aggregation rate in vitro was induced by adenosine diphosphate (ADP) and collagen, respectively. The effects of DWSTB on the platelet aggregation rate were investigated in vivo and in vitro, respectively. Results: The maximum in vivo platelet aggregation rate in the rat was significantly decreased after administration of 2.0 and 4.0 g·kg-1 DWSTB (P<0.05.P<0.01). The maximum rabbit platelet aggregation rate induced by ADP and collagen in vitro were suppressed significantly by 2.0-16.0 mg·mL-1 and 2.0-8.0 mg·mL-1 DWSTB, respectively (P<0.05.P<0.01). And the effect of DWSTB on platelet aggregation was raised with increase ofits dose. Conclusion: Dan Wei Powder Tea Bag can restrain the aggregation of platelet in vivo and in vitro.
