Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein(RNP)electroporation

Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Knockout of TMEM206 in mice associated with a loss of corneal transparency

AIM:To investigate the role of transmembrane protein 206(TMEM206)in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing technology.METHODS:TMEM206-knockout mice were generated using the CRISPR-Cas9 system.Variations in ophthalmic pathology were observed using slit lamp microscope and optical coherence tomography(OCT),intraocular pressure(IOP)was measured using a TonoLab Rebound Tonometer,and the ultrastructure of the corneal was observed using a transmission electron microscope.RESULTS:Corneal opacity was observed in 4/18 homozygous TMEM206^(-/-)mice whereas a similar change was not observed in heterozygous TMEM206^(+/-)mice and wild-type littermates.OCT examination showed that the mean central cornea thickness was 125±5.4μm in 4 homozygous TMEM206^(-/-)mice developed corneal edema and 115±1.2μm in wild-type mice(t=3.468,P<0.05)at 43wk.The mean IOP was 12.08±0.07 mm Hg in four right eyes with corneal edema and 12.03±0.03 mm Hg in three normal left eyes(P>0.05).Transmission electron microscopy revealed a disruption in the organization of the collagen fibrils in the central part of the cornea in homozygous TMEM206^(-/-)mice.CONCLUSION:TMEM206 is associated with corneal edema which caused organizational disruption of collagen fibrils in corneas of mice.

Motor neuron-specific RhoA knockout delays degeneration and promotes regeneration of dendrites in spinal ventral horn after brachial plexus injury

Dendrites play irreplaceable roles in the nerve conduction pathway and are vulnerable to various insults.Peripheral axotomy of motor neurons results in the retraction of dendritic arbors,and the dendritic arbor can be re-expanded when reinnervation is allowed.RhoA is a target that regulates the cytoskeleton and promotes neuronal survival and axon regeneration.However,the role of RhoA in dendrite degeneration and regeneration is unknown.In this study,we explored the potential role of RhoA in dendrites.A line of motor neuronal conditional knockout mice was developed by crossbreeding HB9~(Cre+)mice with RhoA~(flox/flox)mice.We established two models for assaying dendrite degeneration and regeneration,in which the brachial plexus was transection or crush injured,respectively.We found that at 28 days after brachial plexus transection,the density,complexity,and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice were slightly decreased compared with that in Cre mice.Dendrites underwent degeneration at 7 and 14 days after brachial plexus transection and recovered at 28–56 days.The density,complexity,and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice recovered compared with results in Cre mice.These findings suggest that RhoA knockout in motor neurons attenuates dendrite degeneration and promotes dendrite regeneration after peripheral nerve injury.

Truncating PICK1 Variant Identified in Azoospermia Affected Mitochondrial Dysfunction in Knockout Mice

Objective The protein interacting with C kinase 1(PICK1)plays a critical role in vesicle trafficking,and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome,which eventually disrupts acrosome formation and leads to male infertility.Methods An azoospermia sample was filtered,and the laboratory detection and clinical phenotype indicated typical azoospermia in the patient.We sequenced all of the exons in the PICK1 gene and found that there was a novel homozygous variant in the PICK1 gene,c.364delA(p.Lys122SerfsX8),and this protein structure truncating variant seriously affected the biological function.Then we constructed a PICK1 knockout mouse model using clustered regularly interspaced short palindromic repeat cutting technology(CRISPRc).Results The sperm from PICK1 knockout mice showed acrosome and nucleus abnormalities,as well as dysfunctional mitochondrial sheath formation.Both the total sperm and motility sperm counts were decreased in the PICK1 knockout mice compared to wild-type mice.Moreover,the mitochondrial dysfunction was verified in the mice.These defects in the male PICK1 knockout mice may have eventually led to complete infertility.Conclusion The c.364delA novel variant in the PICK1 gene associated with clinical infertility,and pathogenic variants in the PICK1 may cause azoospermia or asthenospermia by impairing mitochondrial function in both mice and humans.

Lycium ruthenicum Murr. treatment attenuates APP_(SWE)/PS1ΔE9 mouse model-like mitochondrial dysfunction in Slc25a46 knockout mouse model

Mitochondrial dysfunction is proposed to be substantially associated with ageing and ageing-related diseases like Alzheimer's disease(AD). However, it is unclear whether different mouse models with mitochondrialrelated diseases have similar changes in mitochondrial morphology of the same tissues. Moreover, whether similarities in mitochondrial morphology can be a suitable marker for screening and/or discovering mitochondrial-protective substances remains unknown. Mitochondria morphology in different tissues of a novel mitochondrial outer membrane protein Slc25a46 knockout mouse and a traditional APP_(SWE)/PS1ΔE9 transgenic mouse were examined using transmission electron microscope(TEM). Both young Slc25a46 knockout mice and aged APP_(SWE)/PS1ΔE9 mice models showed similar mitochondrial damage in cerebellum tissues. The results indicated that different mitochondrial-related diseases shared similar alteration and defects in mitochondrial morphology. Furthermore, Lycium ruthenicum Murr. extract, a bioactive food substance with cognition-improving property, could effectively improve muscle strength and increase body weight in the Slc25a46 knockout mice. These findings suggest that mitochondrial morphology defects in mice models, particularly in the mitochondrial compartment, represent a unified and effective marker for screening and validating natural product-derived functional substances with mitochondrial protective properties. It also holds potential application in mitochondrial-impaired senile neurodegenerative diseases, especially in AD.

Knockout血清替代品可提高C57BL/6J小鼠胚胎干细胞建系效率

目的:在培养液中添加knockout血清替代品(knockout serum replacement,KSR)代替胎牛血清(FBS)用于建立C57BL/6J小鼠胚胎干细胞(ESC)细胞系,以便消除血清中的不确定因子对ESC增殖的影响。方法:以C57BL/6J小鼠3.5 d的囊胚为材料分离ESC,比较KSR和FBS用于建立小鼠ESC细胞系的效率,并通过体内、外分化验证所分离获得的小鼠ESC的发育潜能。结果:培养液中添加KSR,成功从13个小鼠囊胚中分离获得一个ESC细胞系(MES-1),体外培养传代超过20代仍保持未分化状态,核型为正常XX型,碱性磷酸酶及oct-4基因高表达,悬浮培养可以生成拟胚体,接种到裸鼠皮下可形成畸胎瘤,注射到ICR小鼠3.5 d囊胚中,ESC可以参与胚胎发育并产生嵌合体小鼠。而培养液中添加FBS的对照组未能获得超过3代的ESC细胞系。结论:在培养液中添加KSR代替FBS适合于C57BL/6J小鼠ESC的分离与培养,从而可避免实验前对所用血清的筛选。

Study on the Knockout and the Soluble Prokaryotic Expression of VP5 Protein Transmembrane Region of IBDV

[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.

BALB/c-HSF_1 Knockout小鼠的主要脏器重量、脏器系数及主要血液生化指标的测定

目的 提供HSF1 Knockout小鼠的脏器重量 ,脏器系数的生物学特性指标。方法 选用成年HSF1Knockout小鼠 5 0只 (雄性 2 1只 ,雌性 2 9只 ) ,分别测定体重和 8个主要脏器重量 ,计算脏器系数 ,测定其主要血液生化指标 ,并对雌雄鼠脏器重量 ,脏器系数进行比较 ,对血液生化指标进行统计。结果 雌雄鼠脾脏系数、肾脏系数差异有显著性 (P <0 0 5 ) ,胃系数、脑系数差异有显著性 (P <0 0 1) ,心脏、肺系数差异不显著 (P >0 0 1)。结论 应注意HSF1 Knockout小鼠实验时的雌雄鼠胃系数、脑系数的显著性差异 ;脾脏系数、肾脏系数的明显差异。

Knockout^(TM) SR无血清培养基支持小鼠精原干细胞的短期存活(简报)

精原干细胞（spermatogonial stem cells，SSCs）是睾丸内具有自我复制和分化为精子潜能的干细胞，它的体外培养是精子发生机理研究和制作转基因动物等的新途径。近几年的研究表明，SSCs在体外的自我增殖需要GDNF（glial cell line—derived neurotrophic factor）因子和饲养层细胞等的支持，并且睾丸支持细胞（Sertoli’s cells）和血清都导致培养的SSCs分化。因此，使用无血清培养基培养高度纯化的SSCs是培养成败的关键之一。

组织特异性KNOCKOUT小鼠──基因打靶技术的新突破

组织特异性ＫＮＯＣＫＯＵＴ小鼠──基因打靶技术的新突破姜宇飞（北京医科大学免疫系北京１０００８３）近十年来，分子生物学研究得到了迅猛的发展。转基因技术及基因Ｋｎｏｃｋｏｕｔ技术为人们在动物体内分析某一特定基因的功能提供了有力的手段。转基因技术是导入一...

Photoshop外挂滤镜KnockOut的应用

Photoshop外挂滤镜除了可以制作出很炫的特效,Knock Out外挂滤镜还可以进行精细抠图,介绍了Knock Out的基本操作,通过三个实例分析了Knock Out的应用过程。

浅谈使用KnockOut工具抠出毛发

抠图是图像处理中常做的操作之一，Photoshop中有很多工具都可以实现抠图，但操作步骤过于复杂，不易掌握，本文主要介绍了使用KnockOut工具抠图的方法，对于常见的毛发抠图进行简单化的抠图处理．

Molecular mechanisms of liver ischemia reperfusion injury:Insights from transgenic knockout models

Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor grafts.The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis.Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury.IRI involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways.Transgenic animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies.Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein.This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.

Knockout mouse models of insulin signaling: Relevance past and future

Insulin resistance is a hallmark of type 2 diabetes. In an effort to understand and treat this condition, re searchers have used genetic manipulation of mice to uncover insulin signaling pathways and determine the effects of their perturbation. After decades of research much has been learned, but the pathophysiology o insulin resistance in human diabetes remains contro versial, and treating insulin resistance remains a chal lenge. This review will discuss limitations of mouse models lacking select insulin signaling molecule genes In the most influential mouse models, glucose metabo lism differs from that of humans at the cellular, organ and whole-organism levels, and these differences limi the relevance and benefit of the mouse models both in terms of mechanistic investigations and therapeutic development. These differences are due partly to im mutable differences in mouse and human biology, and partly to the failure of genetic modifications to produce an accurate model of human diabetes. Several fac tors often limit the mechanistic insights gained from experimental mice to the particular species and strain including: developmental effects, unexpected meta bolic adjustments, genetic background effects, and technical issues. We conclude that the limitations and weaknesses of genetically modified mouse models of insulin resistance underscore the need for redirection of research efforts toward methods that are more directly relevant to human physiology.

Human androgen deficiency： insights gained from androgen receptor knockout mouse models

The mechanism of androgen action is complex. Recently, significant advances have been made into our understanding of how androgens act via the androgen receptor （AR） through the use of genetically modified mouse models. A number of global and tissue-specific AR knockout （ARKO） models have been generated using the Cre-loxP system which allows tissue- and/or cell-specific deletion. These ARKO models have examined a number of sites of androgen action including the cardiovascular system, the immune and hemopoetic system, bone, muscle, adipose tissue, the prostate and the brain. This review focuses on the insights that have been gained into human androgen deficiency through the use of ARKO mouse models at each of these sites of action, and highlights the strengths and limitations of these Cre-loxP mouse models that should be considered to ensure accurate interpretation of the phenotype.

Effects of p75 neurotrophin receptor knockout on axonal regeneration in a mouse model of facial nerve injury

BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.

High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.

Localization of NKCC1 in the Cochlea and Morphology of the Cochlea in NKCC1-Knockout Mice

The distribution of the Na-K-2Cl co-transporter （NKCCl） in the cochlear K^＋ cycling pathway in cochlea and cochlear histological changes in the NKCCl knockout mice were investigated. By using immunohistochemistry and toluidine blue staining, the localization of NKCCl in cochlea of the C57BL/6J mice and the cochlear histological changes in the NKCCl knockout mice were observed. It was found that the NKCCl was expressed mainly in the stria marginal cells and the fibrocytes in the inferior portion of the spiral ligament in the adult C57BL/6J mice. Subpopulation of the fibrocytes in the suprastrial region and the limbus was also moderately immunoreactive. While in the cochlea of the NKCCl knockout mice, Reissner＇s membrane was collapsed and scala media disappeared, accompanied with the loss of inner hair cells, outer hair cells and the support cells. The tunnel of Corti was often absent. All the findings suggested the localization of NKCCl in the cochlea was closely correlated with cochlear K^＋ cycling. Loss of NKCCl led to the destruction of the cochlear structures, and subsequently influenced the physiological function of cochlea.

Reduced prostate branching morphogenes,s in stromal fibroblast, but not in epithelial, estrogen receptor a knockout mice

Early studies suggested that estrogen receptor alpha （ERa） is involved in estrogen-mediated imprinting effects in prostate development. We recently reported a more complete ERa knockout （KO） mouse model via mating β-actin Cre transgenic mice with floxed ERa mice. These ACTB-ERaKO male mice showed defects in prostatic branching morphogenesis, which demonstrates that ERa is necessary to maintain proliferative events in the prostate. However, within which prostate cell type ERa exerts those important functions remains to be elucidated. To address this, we have bred floxed ERa mice with either fibroblast-specific protein （FSP）-Cre or probasin-Cre transgenic mice to generate a mouse model that has deleted ERa gene in either stromal fibroblast （FSP-ERaKO） or epithelial （pes-ERaKO） prostate cells. We found that circulating testosterone and fertility were not altered in FSP.ERaKO and pes-ERaKO male mice. Prostates of FSP-ERaKO mice have less branching morphogenesis compared to that of wild-type littermates. Further analyses indicated that loss of stromal ERa leads to increased stromal apoptosis, reduced expression of insulin-likegrowth factor-1 （IGF-1） and FGFIO, and increased expression of BMP4. Collectively, we have established the first in vivo prostate stromal and epithelial selective ERaKO mouse models and the results from these mice indicated that stromal fibroblast ERa plays important roles in prostatic branching morphogenesis via a paracrine fashion. Selective deletion of the ERa gene in mouse prostate epithelial cells by probasin-Cre does not affect the regular prostate development and homeostasis.

Claudin-7 gene knockout causes destruction of intestinal structure and animal death in mice

BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system.AIM To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout mice.METHODS We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers.RESULTS We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional Claudin-7 knockout mice was increased compared with control mice(small intestine: 54.1 ± 2.96 vs 38.4 ± 1.63;large intestine: 44.7 ± 1.93 vs 27.4 ± 0.60; P < 0.001).CONCLUSION The knockout of Claudin-7 in vivo causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice.Claudin-7 may act as a tumor suppressor gene in the development of colorectal cancer.