利用CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入研究

为研究CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入的可行性,将包装不同滴度增强型绿色荧光报道基因(Enhance green fluorescent protein,EGFP)的腺病毒载体和慢病毒载体显微注射到HH14时期鸡胚的外周血管中,对胚胎发育至3.5 d和9d鸡胚存活、各器官中EGFP荧光强度等指标进行...     

鸡X期胚盘细胞电转染外源基因条件的探索

以EGFP为报告基因体外电穿孔转染鸡X期胚盘细胞,探讨适宜电转染条件以及影响鸡ES细胞存活率和转染率因素。分离鸡X期胚盘细胞进行培养、传代及鉴定,二代细胞设定不同的电压、脉冲时间、质粒浓度、细胞密度及孵育温度等条件转染。电击后10 min,0.4%台盼蓝染色,血细胞计数板计数,计算存活率。48 h后在荧光显微镜下计绿色荧光细胞数和总细胞数,计算转染率。结果表明:电转染鸡ES细胞的最优化电压为280 v,脉冲时间为75μs,质粒浓度为20μg/mL,转染前4℃、转染后25℃(室温)或4℃各静置10 min,细胞密度调整在1×105个/mL×106个/mL,尽量使细胞处于对数生长期。电穿孔法转染鸡ES细胞是简便有效的方法,利用优化条件转染ES细胞,可以满足进一步实验的要求。     

双基因共表达的"自杀性"DNA疫苗载体的改造及其体外表达特性

PCR扩增西门利克森林病毒（SFV）亚基因组启动子26S序列及人工合成Linker，将其插入基于SFV复制子的“自杀性”DNA疫苗表达载体pSCA1的多克隆位点，获得舍2个26S启动子并能同时驱动双基因共表达的“自杀性”DNA疫苗表达载体pSCA2。为了验证pSCA2的表达能力，PCR扩增绿色荧光蛋白基因EGFP和红色荧光蛋白基因DsRed2，分别插入pSCA2的2个26s启动子下游，获得EGFP和DsRed2双基因共表达的“自杀性”DNA疫苗pSCA2-EGFP／RFP。将pSCA2-EGFP／RFP转染BHK-21细胞，转染后24h，可同时观察到被转染细胞发绿色荧光和红色荧光，证实EGFP和DsRed2均获得表达。     

Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Gene delivery in the equine cornea: a novel therapeutic strategy

Objective To determine if hybrid adeno‐associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene, and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Material Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO₂. Procedure AAV5 vector expressing EGFP under control of hybrid cytomegalovirus + chicken β‐actin promoter was applied topically to ECF. Expression of delivered EGFP gene in ECF was quantified using fluorescent microscopy. Using fluorescent staining, the total number of cells and transduction efficiency of tested AAV vector was determined. Phase contrast microscopy, trypan blue and TUNEL assays were used to determine toxicity and safety of AAV5 for ECFs. Results Topical AAV5 application successfully transduced significant numbers of ECFs. Transduction efficiency was 13.1%. Tested AAV5 vector did not cause phenotype change or significant cell death and cell viability was maintained. Conclusions Tested AAV5 vector is effective and safe for gene therapy in ECFs in vitro.     

ZB transposon and chicken vasa homologue (Cvh) promoter interact to increase transfection efficiency of primordial germ cells in vivo

ABSTRACT

1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter.

2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2–3 or via blood vessel at HH stage 13–14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13–14 were significantly higher than that at HH stage 2–3.

3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence.

4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13–14 may be a more efficient route for PGCs transfection in vivo.     

Influence of Macrophages on the Rooster Spermatozoa Quality

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low‐density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer‐assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2–3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10–15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p ? 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy.     

Genetic Parameters for Semen Traits in AI Boars Estimated from Data on Individual Ejaculates

Semen volume, sperm concentration, percentage progressive motility, percentage of abnormal spermatozoa and total number of sperms from 215 830 ejaculates were analysed. The ejaculates were collected between 2000 and 2005 and originated from 3675 boars of different breeds and crossbred combinations from 23 AI centres of the Czech Republic. Genetic parameters were estimated using multiple- and single-trait animal models. Factors included in the models were: breed or crossbred combination, year–month, age class of the boar, interval between subsequent semen collections, joint effect of AI centre and year, permanent environment and additive genetic effect of the boar. The estimated heritabilities for semen volume and sperm concentration were approximately 0.20, whereas the estimates were somewhat lower for motility, percentage of abnormal sperm and total number of sperms. High negative genetic correlations were estimated between semen volume and sperm concentration (−0.68 for dam breeds, −0.69 for sire breeds) and between motility and percentage of abnormal sperm (−0.93 for dam breeds and −0.59 for sire breeds). The correlations between both semen volume or sperm concentration and motility or percentage of abnormal sperm were mostly small and negligible. Repeatabilities of 0.43–0.46, 0.37–0.38, 0.29–0.35, 0.42–0.50 and 0.29–0.30 were estimated for semen volume, sperm concentration, motility, percentage of abnormal sperm and the total number of sperms, respectively. On the basis of the estimated genetic parameters presented here, effective selection on sperm characteristics, especially for volume and concentration, should be possible using an animal model.     

Distribution and Expression of Recombinant Plasmid Encoding Chicken Interleukin-2

A plasmid DNA that encodes chicken interleukin-2 (pCI-ChIL-2-EGFP) was investigated for its distribution and expression after intramuscular (i.m.) injection in chickens. After the i.m. injection, serum distribution was detectable from 2 h post inoculation (p.i.), peaked at 8 h p.i., and disappeared at 7 days p.i. The plasmid DNA was also observed in several organs including heart, liver, lung, spleen, bursa and inoculated muscle at different time points, but at 19 days p.i. the plasmid DNA was not found in any organ except inoculated muscle. Fluorescence of enhanced green fluorescent protein (EGFP) was found in cytoplasm and nucleus of cultured Vero cells, chicken embryo fibroblasts and peripheral blood lymphocytes, which were transfected in vitro with the plasmid DNA or in vivo with Lipofectamine. The expression profile of the fusion gene (ChIL-2-EGFP) in vivo was measured by RT-PCR, ELISA and fluorescence microscopy. The EGFP expression was detected from 8 h p.i. to 14 days p.i. and peaked at 5 days p.i., when the number of EGFP-expression myocytes was about 5% in the injected site. These results demonstrate that intramuscular administration of plasmid DNA leads to widespread distribution and long-term expression in vivo.     

Cryodamage of plasma membrane and acrosome region in chicken sperm

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil⁺ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.     

鸡胚精原细胞体外转染EGFP方法的比较

以增强型绿色荧光蛋白（Enhanced green fluorescent protein,EGFP）为报告基因体外转染鸡胚胎精原细胞（Chicken embryonic spermatogonial cells,CESCs）,比较磷酸钙、脂质体（梭华-Sofast）、电穿孔3种方法转染EGFP的效率,以获得高效的体外转染方法。本研究分离孵化16d的鸡胚睾丸,获取CESCs,体外培养和传代扩增,传至第2代后进行碱性磷酸酶（AKP）活性和阶段特异性表面抗原-1（Stage specific embryonic antigen-1,SSEA-1）免疫荧光鉴定。运用磷酸钙、脂质体（梭华-Sofast）、电穿孔3种方法体外转染第2代CESCs,荧光细胞计数法分析转染效率。结果表明：与磷酸钙法、脂质体法相比,电穿孔法可获得更高的细胞成活率和转染率（68％ vs 39％、65％,19．70％ vs 2．92％、9．73％）,差异显著（P〈0．05）。因此得出,电穿孔法是将外源基因导入鸡胚精原细胞的适宜方法。     

Neurogenic Differentiation of EGFP Gene Transfected Amniotic Fluid‐Derived Stem Cells from Pigs at Intermediate and Late Gestational Ages

The aims of this study were (i) to determine whether amniotic fluid‐derived stem cells (amniotic fluid‐derived stem; AFS cells) could be isolated from pigs at intermediate and late gestational ages, and (ii) to determine if these AFS cells could be differentiated in vitro into neural lineages following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Amniotic fluid‐derived stem cells were isolated from embryonic day 60 and day 110 porcine amniotic fluid respectively, and transfected with EGFP gene using lipofection. The transfected AFS cells were induced to differentiate into cells of neuronal lineages. Markers associated with undifferentiated AFS cells and their neural derivatives were tested by polymerase chain reaction. The results demonstrated that porcine AFS cells could be isolated at intermediate and late gestational ages and that transfected AFS expressed EGFP and could be induced to differentiate in vitro. Undifferentiated AFS cells expressed POU5F1, THY1 and SOX2, while following differentiation cells expressed markers for astrocytes (GFAP), oligodendrocytes (GALC) and neurons (NF, ENOS and MAP2).     

Kisspeptin injection improved the semen characteristics and sperm rheotaxis in Ossimi ram

The aim of the present study was to investigate the effect of kisspeptin-10 (Kp10) injection on semen characteristics, testosterone (T) production and sperm rheotaxis using microfluidic devices in immature ram. Computer-assisted sperm analysis (CASA) with controlled flow velocity was used to explore the kinetic parameters of sperm and positive rheotaxis (PR %). PR % was defined as the number of PR sperms over the number of motile sperms. Healthy Ossimi rams were randomly divided into two groups; a saline-treated control group and Kp10-treated one (5 µg/kg body weight). Treatments were given by intramuscular injection once a week for 1 month. After 1 month, the semen was collected and evaluated weekly for 6 weeks, while the blood samples were collected every 2 weeks for the next 8 weeks. Semen properties were significantly affected by Kp10 injection (p < .01). The Kp10 increased the volume, sperm concentration and percentages of live sperm compared with those of control. Additionally, sperm trajectories and rheotaxis get improved by the injection of Kp10 with time. Furthermore, kisspeptin improved the secretion of testosterone levels throughout the period of study. In conclusion, injections of the Kp10 had a positive impact on semen characteristics as well as improved sperm rheotaxis of Ossimi rams in subtropics.     

荧光探针JC-1与PI联合染色法在猪精子线粒体膜电位检测中的应用

目前,对于精子线粒体膜电位(MMP)的检测分析,通常采用JC-1单独染色,这种染色方法可以准确地区分高、低MMP精子,但不能明确区分精子的存活状态。本研究采用JC-1与碘化丙啶(propidium iodide,PI)2种荧光染料联合染色的方法,对6种不同保存条件下的猪精子进行染色,分别使用流式细胞仪与荧光显微镜2种检测手段对猪精子MMP进行检测。结果表明:死亡精子头部呈红色,高MMP精子尾部呈橙红色,低MMP精子尾部为绿色。2种检测手段的结果比较显示,荧光显微镜能够有效地区分出高、低MMP死精子,但对于活精子MMP的高低检测效果不甚理想。相反,流式细胞仪则能够有效地区分活精子MMP的高低,但对于死精子MMP高低的检测仍较困难。因此,对于猪精子线粒体MMP的检测,采用JC-1与PI双染的方法,用流式细胞仪与荧光显微镜进行联合检测,能比较全面的反映出精子线粒体的功能状态。     

The evaluation of fertility characteristics in hens using fluorescence microscopy

A method for the assessment of fertility in domestic hens is described. Vitelline membranes of fertilized eggs were coloured with fluorochrome DAPI, following which the DNA of sperms on these membranes has been examined with the help of fluorescence microscopy. After insemination with frozen semen there exist significant correlations between the number of sperms on the vitelline membranes on the one hand and 1) fertilization rates; 2) the length of fertile periods. Furthermore, it was observed that with higher numbers of sperms on the membranes, the length of the fertile periods tends to be shorter than with smaller numbers of sperm.     

Methods of collection,extender type,and freezability of semen collected from creole bulls raised in the tropical highlands of Ecuador

This study was conducted to determine the best combination between two collection method and two extenders in the cryopreservation of semen from creole bulls adapted to highlands of the Ecuadorian Andes. Sixty ejaculates from three adult Creole bulls were evaluated after collection by artificial vagina (AV) and electroejaculation (EE). Semen samples were split into two aliquots and diluted with a soy lecithin extender (Andromed®; A) or an egg yolk-containing extender (Triladyl®; T) and packed in straws of 0.25 ml with 20 × 10⁶ sperms. Optical microscopy and computer-assisted semen analysis system (CASA) were used to evaluate semen quality characteristics. The effects of collection methods and extender type as well as its interaction were evaluated by a factorial ANOVA and Bonferroni’s test. Semen samples collected with EE and frozen with T (EE-T) and A (EE-A) had greater proportion of spermatozoa with optical assessed individual progressive motility (IPM), plasmatic membrane intact (HosT), and lower tail abnormalities than those obtained with AV and frozen with the same extenders (AV-T and AV-A); however, differences were significant only between EE-A and AV-T. CASA assessment indicated that the total mobility (TM) was greater (P < 0.05) in semen samples diluted with T, although these samples had a greater proportion (P < 0.05) of sperms with local motility (LM) and fewer immobile sperms (IS), than those extended with A. Generally, semen samples obtained with EE or AV and diluted with T seems to be the best option to ciopreserve gametes of Creole bulls raised in highlands of Ecuadorian Andes.

     

Canine corneal fibroblast and myofibroblast transduction with AAV5

Objective The aims of this study were (1) to determine the efficacy of adeno‐associated vector serotype 5 (AAV5) for delivering gene therapy to canine corneal fibroblasts (CCFs) and myofibroblasts (CCMs) using enhanced green fluorescent protein (GFP) marker gene and (2) to evaluate the cytotoxicity of AAV5 to CCFs and CCMs using an in vitro model. Methods Healthy donor canine corneas were used to generate primary CCFs by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum‐free medium containing transforming growth factor β1 (1 ng/mL). An AAV5 titer (6.5 × 10¹² μg/mL) expressing GFP under control of hybrid cytomegalovirus + chicken β‐actin promoters (AAV5‐gfp) was used to transduce CCF and CCM cultures. Delivered gene expression in CCFs and CCMs was quantified using immunocytochemistry, fluorescent microscopy, and real‐time PCR. Transduction efficacy of the AAV5 vector was determined by counting DAPI‐stained nuclei and EGFP‐positive cells in culture. Phase‐contrast microscopy, trypan blue, and dUTP nick end labeling (TUNEL) assays were used to determine the toxicity and safety of AAV5 in this canine corneal model. Results Topical AAV5 application successfully transduced a significant population of CCFs (42.8%; P < 0.01) and CCMs (28%; P < 0.01). Tested AAV5 did not affect CCF or CCM phenotype or cellular viability and did not cause significant cell death. Conclusions The tested AAV5 is an effective and safe vector for canine corneal gene therapy in this in vitro model. In vivo studies are warranted.     

Secondary sexual traits and semen characteristic of chicken germline chimeras

Birds obtained by embryo engineering are used to study embryo development and to produce transgenic birds. As this method of producing birds still generate strong emotions of the public opinion head ornaments, testes and semen characteristics of sex chimera roosters were examined to check whether they differ from chickens obtained by non‐manipulated methods. Measurements of head ornaments, testes and semen were correlated with each other. Semen quality factor (SQF) was calculated, as well as the level of fluctuating asymmetry (FA) of bilateral traits (wattles and testes). Positive correlation was found for comb width and wattle length and comb thickness and sperm concentration. Semen characteristics and FA did not exceed the level encounter in other chicken lines. Results obtained indicate that germline chimeras are similar in appearance of secondary sexual traits, and semen and testes characteristics to chickens produced in non‐manipulated way.