Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
Animals ; Base Sequence ; Cloning, Organism ; Electrophoresis ; Endonucleases ; genetics ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Gene Knockout Techniques ; Gene Targeting ; methods ; Goats ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Zinc Fingers

Objective: To study the correlation between angiotensin II type1 receptor autoantibodies (AT1-AAs) and the onset risk in patients with essential hypertension (EH) combiningacute coronary syndrome (ACS). Methods: Our research included in 4 groups: EH+ACS group,n=28, ACS group, n=29, EH group,n=26 and Control group,n=23 normal subjects. Serum levels of AT1-AAs were examined by ELISA; high sensitivity C-reactive protein (hs-CRP), blood levels of lipids and glucose were also measured and compared among different groups. The correlations between AT1-AAs and blood lipids, glucose, hs-CRP were studied by multiple linear regression analysis. Results: Compared with Control group, ACS group had similar level of AT1-AAs (0.26±0.09) vs (0.21±0.06),P=0.105; while EH+ACS group and HE group had increased AT1-AAs level as (0.40±0.005) and (0.33±0.10),P=0.001 andP=0.02 respectively; AT1-AAs level was higher in EH+ACS group than HE group,P=0.044. In addition, serum AT1-AAs level was positively related to hs-CRP in EH+ACS group, ACS group and EH group (r=0.589,r=0.503 andr=0.273, allP<0.01). Conclusion: Serum AT1-AAs level was positively related to the onset risk in patients with EH combining ACS; AT1-AAs was also related to hs-CRP at certain degree.

ObjectiveTo explore the clinical characteristics and radiological manifestations of immune reconstitution inflammatory syndrome(IRIS)in acquired immunodeficiency syndrome(AIDS)patients with tuberculosis(TB)during highly active antiretroviral therapy(HAART).MethodsThe clinical and radiological data in 4 AIDS patients with TB who presented IRIS were analyzed retrospectively.ResultsThe clinical presentations of IRIS in 4 patients included fever(4 cases),weakness and weight loss(3 cases),abdominal pain(2 cases),cough with sputum(1 case),dyspnea(1 case).Cervical and(or)supra-clavicular lymph node enlargement were seen in 3 patients,inguinal lymph node enlargement in 1 patient,abdominal lymph node enlargement in 1 patient,hilar or mediastinal lymph node enlargement in 2 patients,pulmonary parenchyma and liver were involved in 2 patients,the involvement of kidney,adrenal gland,mesentery,peritoneum,psoas,brain and cutis was respectively found in 1 patient.The clinical and radiological presentations of IRIS were temporary and self-limited,improvement can be seen with antituberculosis therapy and HAART. ConclusionsIt is possible to have IRIS during HAART in AIDS patients with TB.Imaging examinations play an important role in the early diagnosis,monitoting and evaluating the response to therapy of IRIS.

Objective To study the characteristics of the transcranial electric stimulation(TES)-elicited motor evoked potentials(MEP)recorded from different points of the diaphragm,and to determine the optimal record- ing site in the diaphragm.Methods One EEG electrode was placed subcutaneously in the midline of the skull as stimulation electrode and another EEG electrode was inserted into hard palate submucously as cathode in 30 male rats.And single square wave electrical pulse was used for stimulation.The stimulation intensity was 15 mA,the du- ration of the pulse was 0.2 ms,and the stimulating interval was 200 ms.The concentric needle electrode were insert- ed into the following eight sites:the medial,intermediate,lateral crura of the diaphragm,the junction of the posterior axilla line and the inferior border of the eleventh rib,the junction of the anterior axilla line and the inferior border of the ninth rib,the junction of the intermediate line of the clavicle and the inferior border of the eighth rib,the junction of the para-sternal line and the inferior border of the seventh rib,the pars sternalis.The MEP was recorded from each point,respectively.The optimal sites of the diaphragm MEP were found and recorded.Results The MEPs were re- corded from each of the 8 sites of the diaphragm in all the rats after TES.There was no statistically significant differ- ence among the latencies of every site.But the amplitude varied from 6.08?0.46 mV in maximum along the inferior border of the ninth rib at the anterior line of the axilla to the minimum of O.91?0.03 mV at the pars sternalis. Conclusion It is safer to insert the needle along the inferior border of the ninth rib at the anterior line of the axilla, because there was certain distant from the folding part of the pleura and the amplitude of the recorded wave at this point is maximal.

The kinetics of nicotine degradation by O.intermedium DN2 and its application in tobacco waste were investigated. The results showed that the optimum temperature of nicotine degradation by O.intermedium was 30 ℃, the pH value was 6.5 and a mount of inoculum was 5 %. Under above conditions, the kinetics of nicotine degradation of initial concentration 500 mg/L was studied. The results indicated that the degradation process of nicotine with no-induced strain DN2 followed inverse S-shaped curve, and degradation process of nicotine with induced cells of DN2 followed Eckenfelder mode. The half life of nicotine degradation was 17.43 h and 4.10 h, respectively. And the results also showed that tolerance of O.intermedium DN2 to nicotine was up to 5000 mg/L when 0.1 % of glucose was added. Nicotine (2 220 mg/L) in extract of tobacco wastes degraded about 95.22 % by strain DN2 in 60 h incubation, indicating that strain DN2 was of application value in treatment nicotine pollution.

Objective To assess the accuracy of neuronavigation of 3D DSA and to evaluate the feasibility of 3D DSA neuronavigational neurosurgery through the coregistration of 3D DSA and MRI(A). Methods A Peg-Board Phantom was used in our study.The phantom consisted of 32 rods which were used for target localization;the height and the location of the rods were in normal distribution.For 3D DSA (Infinix NS/VC,Toshiba),the raw data was reconstructed to 3D images on the DSA workstation,and transferred to a online PC workstation where it was converted to standard 2D DICOM image data using WFU DICOM_Toolkit software.For MRI(A),the phantom was scanned with FSPGR sequence on the MRI scanner(GE SigMa VH/i 3.0 T),and the DICOM images were also transferred to the online PC workstation.Using the software 3D Slicer registration was performed on the PC workstation by using the location and shape of the rods in the phantom.The localization error of the rods was measured in image space as the Euclidean distance between targets defined in image space and those detected in the physical space. Paired t test was used to evaluate the difference between the accuracy of neuronaviagtion of 3D DSA and that of MRI(A).Results Through the coregistration of the rods in the phantom from different modality,all the images were better coregistrated.The mean localization error was(0.38?0.24)mm(3D DSA)and (0.31?0.12)mm[MRI(A)].There was no significant statistical difference between the accuracy of neuronavigation of 3D DSA and MRI(A)(t=-0.601,P=0.55).Conclusion 3D DSA images can be used in the neuronavigation system through the coregistration of 3D DSA and MRI(A).

Objective To evaluate the clinical efficacy of multiple stents placement in the management of hilar cholangiocarcinoma,especially in the complex cases of which the hepatic ducts are invaded.Methods Forty-five consecutive patients with hilar cholangiocarcinoma were treated with percutaneous transhepatic placement of two or three self-expandable metallic endoprostheses.The cause of hilar obstructions in these patients were all cholangiocarcinoma,including Bismuth classification type Ⅱ(n 12 ),Ⅲa(n 17),Ⅲb(n 10),and Ⅳ(n 6).Two or 3 stents were placed in the configuration of T,Y or X over the strictures.Results Stent placement with 2 or 3 endoprostheses was successful in all patients.All patients showed significant decrease in serum bilirubin level.The mortality rate within 30 days of stent placement was 2.2%(1/45).The mean survival and stent patency times were 215.3 d(26— 516 d)and 181.5 d(26—473 d),respectively.Conclusion Deploying of multiple metallic stents is an effective method to treat complex hilar cholangiocarcinoma,especially for the cases of which hepatic ducts are invaded:the henatic ducts should be drained as much as nossible.

Objective To summarize the experience of the therapy and diagnosis of thoracictuberculosis. Methods Diagnosis and operation of 163 cases of thoracictuberculosis were analyzed.Results 163 cases of thoracic- tuberculosis were treated with focuspurge upon two weeks' anti-tuberculosis treatment.153 cases were cured upon one operation.10 cases suffered incision delayed healing and there were no recurrence cases.Conclusion Thoraeictu- berculosis was treated with focuspurge upon two weeks anti-tuberculosis treatment before operation.Complete purge of focus and postoperative compression band and residual cavity filled with music flap were important measures to prevent incision delayed healing and recurrence.

[Objective] To investigate the clinical characteristics and blood transfusion status of patients of liver cirrhosis and analyze its rationality.[Methods] We designed questionnaires to collect the data of patients admitted with liver cirrhosis including clinical features,blood transfusion,smoking,drinking and other living habits.We follow up the patients and analyze the blood transfusion rationality.[Results] Data on 198 patients was collected.34.8％ (69/198) of all patients were transfused at least one blood component.Total blood transfusion was 371 times,of which 52.2％ of the blood transfusion cases (36/69) were transfused with two or more blood during hospitalization.Among the 69 cases of blood transfusion,11 cases were treated with the first blood transfusion for the purpose of treatment and 58 cases for prevention.18 of those cases were infused with red blood cells of 90.5 units.54.55 ％ (60/110) and 60.91％ (67/110) of patients who had a pre-transfusion INR＞1.3 did not receive plasma.2.27％ (2/88) of patients who had a pre-transfusion INR≤1.3 received plasma.29.41％ (5/17)who had a pre-transfusion fib≤1.0 received cryoprecipitate.3.87％(7/181) who had a pre-transfusion fib＞1.0 received cryoprecipitate.[Conclusions] Blood transfusion is common in patients with liver cirrhosis.Empirical and preventive blood transfusion is common also.We should take a more scientific restrictive blood transfusion strategy.

Objective To investigate the molecular genetic causes and their characteristics of deafness from patients with nonsyndromic hearing loss in Gansu province.Methods Peripheral blood samples were obtained from a total of 375 patients with nonsyndromic hearing loss to extract genomic DNA.Three genes of GJB2, mitochondrial DNA 12SrRNA, and SLC26A4 were screened for mutations in our study cohort using SNPscan technology.Results Among 375 patients, 23 patients were found to carry the homoplasmic mtDNA12SrRNA A1555G mutation, and 2 patients were detected to carry the homoplasmic mtDNA12SrRNA C1494T mutation.Forty-two cases(11.2%) were caused by GJB2 mutations, including 31cases(8.3%) of homozygous mutations, 11 patients(2.9%) of compound heterozygous mutations, and 25 cases(6.7%) of single homozygous mutations.c.235delC was the most prevalent GJB2 mutation with the allele frequency of 8.8%.Twenty-nine cases (7.7%) were caused by SLC26A4mutations, including 17cases(4.5%) of homozygous mutations, 12 patients(3.2%) of compound heterozygous mutations, and 16 cases(4.3%) of single homozygous mutations.c.919-2A>G and c.2168A>G were the most common SLC26A4 mutation, the allele frequencies were 5.2% and 2.0%, respectively.Conclusion A high incidence of mtDNA12SrRNAA1555G mutation is found in nonsyndromic hearing loss patients from Gansu province, while the incidence of GJB2 and SLC26A4 mutations is similar to the level of the overall Chinese deaf population.These findings demonstrate that a total of 25.6% of deaf patients have inherited hearing impairment caused by GJB2, SLC26A4, and mitochondrialDNA12SrRNA mutations.As a result 36% patients and family member can acquire effective genetic counseling.