CRISPR-based genetic control strategies for insect pests

Genetic control strategies such as the sterile insect technique have successfully fought insect pests worldwide. The CRISPR(clustered regularly interspaced short palindromic repeats) technology, together with high-quality genomic resources obtained in more and more species, greatly facilitates the development of novel genetic control insect strains that can be used in area-wide and species-specific pest control programs. Here, we review the research progress towards state-of-art CRISPR-based genetic control strategies, including gene drive, sex ratio distortion, CRISPRengineered genetic sexing strains, and precision-guided sterile insect technique. These strategies’ working mechanisms,potential resistance development mechanisms, and regulations are illustrated and discussed. In addition, recent developments such as stacked and conditional systems are introduced. We envision that the advances in genetic technology will continue to be one of the driving forces for developing the next generation of pest control strategies.

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.

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.

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.

Engineering of Corynebacterium glutamicum to Enhance L-ornithine Production by Gene Knockout and Comparative Proteomic Analysis

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.

Gene knockout or inhibition of macrophage migration inhibitory factor alleviates lipopolysaccharide-induced liver injury via inhibiting inflammatory response

Background:Liver injury is one of the most common complications during sepsis.Macrophage migration inhibitory factor(MIF)is an important proinflammatory cytokine.This study explored the role of MIF in the lipopolysaccharide(LPS)-induced liver injury through genetically manipulated mouse strains.Methods:The model of LPS-induced liver injury was established in wild-type and Mif-knockout C57/BL6 mice.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBil)were detected,and the expressions of MIF,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were measured.Liver histopathology was conducted to assess liver injury.Moreover,the inhibitions of MIF with(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester(ISO-1)and 4-iodo-6-phenylpyrimidine(4-IPP)were used to evaluate their therapeutic potential of liver injury.Results:Compared with wild-type mice,the liver function indices and inflammation factors presented no significant difference in the Mif-/-mice.After 72 h of the LPS-induced liver injury,serum levels of ALT,AST,and TBil as well as TNF-αand IL-1βwere significantly increased,but the knockout of Mif attenuated liver injury and inflammatory response.In liver tissue,m RNA levels of TNF-α,IL-1βand NF-κB p65 were remarkably elevated in LPS-induced liver injury,while the knockout of Mif reduced these levels.Moreover,in LPS-induced liver injury,the inhibitions of MIF with ISO-1 and 4-IPP alleviated liver injury and slightly attenuated inflammatory response.Importantly,compared to mice with LPS-induced liver injury,Mif knockout or MIF inhibitions significantly prolonged the survival of the mice.Conclusions:In LPS-induced liver injury,the knockout of Mif or MIF inhibitions alleviated liver injury and slightly attenuated inflammatory response,thereby prolonged the survival of the mice.Targeting MIF may be an important strategy to protect the liver from injury during sepsis.

Metabolic regulation of <i>Escherichia coli</i>cultivated under anaerobic and aerobic conditions in response to the specific pathway gene knockouts

Effect of the specific gene knockout on the main metabolism in Escherichia coli was reviewed, and the regulation mechanisms were clarified based on different levels of information such as gene expressions, enzyme activities, intracellular metabolite concentrations, and metabolic fluxes together with fermentation data. The effects of the knockout of such genes as pflA, pta, ppc, pykF, adhE, and ldhA on the metabolic changes were analyzed for the case under anaerobic condition. The effects of the knockout of such genes as pgi, zwf, gnd, ppc pck, pyk, and lpdA on the metabolic changes were also analyzed for the case under aerobic condition. The metabolic regulation analysis was made focusing on the roles of transcription factors.

Construction and biological characterization of Burkholderia pseudomallei sRNA gene deletion strain

Objective:Construction of Burkholderia pseudomallei(B.pseudomallei)sRNA knockout strains and observation of their biological function.Methods:Design 9sF/9sR,9xF/9xR and R1/F1 primers,which were used to amplify the homologous arm fragment upstream and downstream of the sRNA gene,through enzyme cutting,ligation,and transformation,the sRNA gene was knocked out from the B.pseudomallei by homologous recombination method.Results:The sRNA mutant was successfully constructed.In comparison with wild strain HNBP001,the growth rate,motility and biofilm formation ofΔsRNA decreased,but the antibiotic sensitivity has no differences.Conclusion:The sRNA knockout strain of B.pseudomallei was successfully constructed,laying a foundation for further research on its mechanism of regulating B.pseudomallei.

Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

Aim： To study polyethylenimine （PEI）-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method. Methods： PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. Results： With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells （especially in primary spermatocytes）. Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis. Conclusion： PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our gtudy, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes. （Asian J Androl 2006 Jan; 8： 53-59）

Genetic Transformation in Triticeae Crops

Wheat, triticale, tritordeum, barley, oat and rye are the most important crops in human consumptions and industry in the world. Transformation technology supplies a new source of improving Triticeae crops. In the past decade, transformation of wheat crops has considerably progressed. Many transgenic plants of Triticeae crops with various genes were produced via nricroprojectile bombardment, Agrobacterium-mediated transformation, PEG-uptake DNA technique, electroporation, microinjection, injection inflorescence and silicone carbide. Integration and expression of transgenes, inheritance and variation of transgenic plants have been studied. Technical improvements of genetic transformation for wheat crops will be extensively useful in commerce and benefit significantly to human being in the world.

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Current gene therapy for stomach carcinoma

Gastric cancer is common in China [1-42],and its early diagnosis and treatment in advanced stage are difficult [31-50].In recent years ,gene study in cancer is a hotspot ,and great progress has been achieved [41-80] .Cancer gene therapy has shifted from the imagination into the laboratory and clinical trials.

Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism

Aim： To investigate the effects of rat Erythropoietin （Epo） on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods： Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups： the first group was given intratesticular injections of pCAGGS-Epo （pCAGGS-Epo group）, the second group was given intratesticular injections of pCAGGS （pCAGGS group）, and the third group were given intratesticular injections of phosphate-buffered saline （PBS group）. At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells （G/S ratio） was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results： The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was （0.85 ± 0.08） g and （0.83 ± 0.03） g, respectively, at week 1 （P = 0.788） and （0.62 ± 0.06） g and （0.52 ± 0.02） g, respectively, at week 2 （P = 0.047）. At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 ± 6.80 vs. 18.63 ± 5.30 at week 1 （P = 0.0078） and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2 （P = 0.1471）, respectively. Conclusion： The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

Intrastriatal Gene Transfer of Vascular Endothelial Growth Factor Rescues Dopaminergic Neurons in a Rat Parkinson's Disease Model

To examine the ability of intrastriatal gene transfer of vascular endothelial growth factor 165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson＇s disease （PD）, we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 （Ad-VEGF）, and injected Ad-VEGF （or Ad-LacZ and PBS as controls） into the striatum of rats 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase （TH） immunohistochemistry were performed to assess the change of dopaminergic neurons. Our results showed that the rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals, It is concluded that intrastriatal gene transfer by Ad-VEGF may rescue the dopaminergic neurons from degeneration in a rat model of PD.

Use of gene-editing technology to introduce targeted modifications in pigs

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.

Animal models of genetic causes of male infertility

Introduction In the past 10 years, more than 100 different genetically engineered mice have been developed with an abnormality in spermatogenesis. More than half of these male infertility animal models have been reported in the past three years alone. Spermatogenic defects in these models vary considerably, ranging from the inappropriate migration of primordial germ cells to the inability of mature spermatozoa to bind the zona pellucida. Many of

Transient Expression of BVDV N^(pro) Gene in vitro and Distribution of Fusion Protein in Cells

The gene encoding nonstructural protein Npro of BVDV was amplified by PCR,then was inserted into pEGFP-C1 vector.The 293 cells were transfected in vitro with recombinant plasmid by liposome.Npro-EGFP fusion protein was viewed directly with fluorensce microscope and mRNA expression of Npro was detected by reverse transcription-polymerase chain reaction（RT-PCR）.Results showed that recombinant plasmid was confirmed to be constructed correctly by PCR,restriction enzyme digestion and DNA sequencing;meanwhile,the gene carried was expressed in 293 cells.The fusion protein had properties of both the two Npro and enhanced green fluorescent proteins（EGFP） and distributed in the cytoplasm.This research lays a foundation for further researches which explored Npro gene function in the process of viral replication and synthesis.

The testis-specifically expressed gene Trim69 is not essential for fertility in mice

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis.The tripartite motif(TRIM)family proteins,most of which have E3 ubiquitin ligase activity,are highly conserved in mammals.They are involved in important cellular processes such as embryonic development,immunity,and fertility.Our previous studies indicated that Trim69,a testis-specific expressed TRIM family gene,potentially participates in the spermatogenesis by mediating testicular cells apoptosis.In this study,we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology.Here,we reported that the male Trim69 knockout mice had normal fertility.The adult knockout mice have shown that the appearance of testes,testis/body weight ratios,testicular histomorphology,and the number and quality of sperm were consistent with wild-type mice.These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility,and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis.This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.

离心涂片法、Gene Xpert技术和改良抗酸染色法用于早期结核性脑膜炎诊断中的临床价值

目的探讨离心涂片法、Gene Xpert技术和改良抗酸染色法应用于早期结核性脑膜炎诊断中的临床价值。方法选择2018年1月至2020年9月就诊于我院的120例脑膜炎患者为研究对象,采用离心涂片法、Gene Xpert技术和改良抗酸染色法对其脑脊液标本中的结核分枝杆菌进行检测。以临床综合诊断结果为金标准,比较三种方法对结核性脑膜炎的诊断价值。结果改良抗酸染色法对结核性脑膜炎的诊断灵敏度、特异度、准确度、阳性预测值、阴性预测值均显著高于离心涂片法、Gene Xpert技术,且Gene Xpert技术显著高于离心涂片法,差异具有统计学意义(P<0.05)。改良抗酸染色法与临床综合诊断结果之间为高度一致性(Kappa值为0.829),Gene Xpert技术与临床综合诊断结果之间为中度一致性(Kappa值为0.634),离心涂片法与临床综合诊断结果之间为低度一致性(Kappa值为0.378)。三种方法对结核性脑膜炎Ⅰ期的诊断符合率从高至低依次为改良抗酸染色法、Gene Xpert技术、离心涂片法,两两比较,差异具有统计学意义(P<0.05);三种方法对结核性脑膜炎Ⅱ期、Ⅲ期的诊断符合率比较,差异无统计学意义(P>0.05)。结论相比于离心涂片法、Gene Xpert技术,改良抗酸染色法可以更加灵敏、准确地诊断早期结核性脑膜炎,值得在临床上推广应用。