大珠母贝精子介导外源基因转移研究

将大珠母贝(PinctadamaximaJameson)精子与“全鱼”GH基因重组体pCAgcGH和pCAgcGHc的线性DNA混合,温育30min,经6次、2⁷、10kV脉冲电处理后,与卵子受精,得到若干贝苗。从贝苗中提取DNA,经PCR扩增和Southernblot分子杂交表明,部分受体带有外源基因,当与精子温育的外源基因浓度分别为2μg/mL,6μg/mL及18μg/mL时,相应贝苗携带外源基因比率分别为56%,20%和50%。即在此范围内,基因转移的阳性率与外源基因的浓度呈正相关。     

精子介导外源DNA转移的研究进展

精子介导外源ＤＮＡ转移的研究进展刘红林陈宜峰（南京师范大学生物系２１００２４）一、引言迄今,为广大研究者所认可的制作转基因动物的基本方法有三种,即ＤＮＡ显微注射法、反转录病毒载体支持的基因转移以及利用转化的全能胚胎干细胞形成生殖系嵌合体的基因转移［１］,然而目前这三种方法制作转基因动物的效率还很低。寻求一种简便、有效、可广泛应用于各种哺乳动物、鸟类、鱼类的转基因方法仍是必要的,精子介导的外源...     

蜜蜂精子介导GFP基因转移研究

江西农业大学动物科技学院郭冬生、天津农学院张巧利和泉州师范学院模式生物研究中心孙亮先三位动物分子生物学研究工作者将pGFP-2质粒线性化,利用精子介导,通过人工授精技术导入意大利蜜蜂处女王．然后将授精王介绍到无王群中繁殖后代,其结果显示：试验群产生了一群绿色荧光阳性蜜蜂,他们对此阳性蜂群后代分析发现,1～2日龄幼虫能坳4到荧光,提取蜜蜂DNA进行PCR扩增到预想的片断。     

电脉冲作用将外源基因导入稀有ju鲫精子的研究

将稀有ju鲫（Gobiocypris rarus）精子与重组质粒pGAhLFc线性DNA混合温育,经电脉冲处理后与卵子受精,孵化出苗。从鱼苗中提取DNA,经PCR检测,25.5%～66.7%鱼苗带有外源基因。在显微镜下观察经电脉冲处理过的精子,发现其活力有不同程度下降,受精率也有不同程度下降,说明不同的电脉冲条件对精子有不同程度的损害作用。精子与外源DNA混合温育,经电脉冲处理后,用DNA外切酶消化后,提取精子DNA,经PCR检测,仍有阳性电泳带,证明电脉冲可以促使稀有ju鲫精子摄入外源基因。     

中华蜜蜂精子介导egfp基因转移

中华蜜蜂Apis cerana cerana是一种真社会性昆虫,也是我国重要的经济昆虫。本实验目的是为了检测精子是否可以作为载体将外源egfp基因介导转入中华蜜蜂。首先将雄蜂精子与线性化的质粒DNA共浴,然后通过人工授精技术将精子导入处女王,再对实验蜂群后代进行分析。结果显示EGFP蛋白在一群实验组蜂的1～2日龄小幼虫中表达较强,能检测到0.01%～0.02%荧光阳性小幼虫个体;通过PCR和RT-PCR技术分析,证实转入的外源egfp基因获得表达。实验结果表明精子载体法能够用于中华蜜蜂外源基因的转移和表达。     

精子介导GFP基因在小鼠早期胚胎中的表达

利用 DIG 末端标记技术和免疫组化技术分析了小鼠精子体外结合内化外源DNA的效率。试验结果表明,不同小鼠个体的精子结合外源DNA的阳性率有明显差异（P<0.01）,平均为13%。利用考马斯亮蓝染色评价了小鼠精子顶体反应发生的情况,筛选出TYH培养液为较合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP基因并获能的小鼠精子与成熟卵母细胞进行体外受精,受精卵进行体外培养,表达GFP胎的阳性率为4.7%。验证了精子介导制备转基因小鼠胚胎的可行性,并建立了利用精子载体法制备转基因小鼠胚胎的平台。     

精子介导GFP基因在小鼠早期胚胎中的表达

利用DIG末端标记技术和免疫组化技术分析了小鼠精子体外结合内化外源DNA的效率。试验结果表明,不同小鼠个体的精子结合外源DNA的阳性率有明显差异(P<0.01),平均为13%。利用考马斯亮蓝染色评价了小鼠精子顶体反应发生的情况,筛选出TYH培养液为较合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP基因并获能的小鼠精子与成熟卵母细胞进行体外受精,受精卵进行体外培养,表达GFP胚胎的阳性率为4.7%。验证了精子介导制备转基因小鼠胚胎的可行性,并建立了利用精子载体法制备转基因小鼠胚胎的平台。     

利用精子介导法向蚕卵导入外源基因的研究

为建立家蚕转基因中切实可行、操作简便的外源基因导入方法,进行了精子介导法探索,以精子介导法的三种方式向家蚕导入所构建质粒pFbGFP,并通过PCR扩增和DNA印迹等手段,已连续两代从基因组DNA检测到导入外源基因GFP的存在,其中的一种导入方式到第二代阳性率约30% .结果表明该法可有效进行家蚕转基因的外源基因导入.     

精子介导鱼类基因转移和聚合酶链反应检测技术

金鱼精子与美洲大绵ｗｅｉ的抗冻蛋白基因一起保温３０分钟后,再与卵子受精,共获得１４５尾成鱼和若干胚胎。从胚胎和成鱼中提取ＤＮＡ经聚合酶链反应（ＰＣＲ法）扩增和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分子杂交表明,外源的抗冻蛋白基因进入了部分受体鱼的染色体组内。测定了４５尾一年龄实验鱼中,有１２尾显示出明确的杂交带,阳性率为２６％。     

外源基因对精子的影响及其在山羊早期胚胎中的表达

摘要: 在前期实验中发现, 山羊精子可自发结合外源DNA, 但结合能力在不同动物个体之间差异显著。挑选结合能力明显不同的3只公羊, 进一步探讨了外源DNA对精子的影响及其在早期胚胎中的表达, 结果发现: 外源基因与精子共同孵育后, 精子的活率、顶体反应发生率和受精能力均呈下降态势, 其降幅与精子的结合能力密切相关。利用与DNA共育后的精子进行体外受精, 外源基因可被导入卵母细胞并在早期胚胎中获得表达, 但胚胎阳性率因精子供体不同而差异显著(P<0.05); 其中来源于高、中结合能力供体生产的胚胎, 分别有16.2%(25/154)和5.3%(4/76)可检测到外源基因存在, 但表达仅见于高结合能力供体生产的早期胚胎, 表达率为6.5%(10/154); 低结合能力供体生产的胚胎无外源基因。研究表明, 在以精子载体方法生产转基因动物的实验过程中, 筛选对DNA结合能力较强的精子供体是提高转基因效率的前提, 但需要考虑外源DNA对精子受精能力的影响。     

电脉冲作用将外源基因导入稀有(鱼句)鲫精子的研究

将稀有鲫 (Gobiocyprisrarus)精子与重组质粒pCAhLFc线性DNA混合温育 ,经电脉冲处理后与卵子受精 ,孵化出苗。从鱼苗中提取DNA ,经PCR检测 ,2 5 .5 %～ 6 6 .7%鱼苗带有外源基因。在显微镜下观察经电脉冲处理过的精子 ,发现其活力有不同程度下降 ,受精率也有不同程度下降 ,说明不同的电脉冲条件对精子有不同程度的损害作用。精子与外源DNA混合温育 ,经电脉冲处理后 ,用DNA外切酶消化后 ,提取精子DNA ,经PCR检测 ,仍有阳性电泳带 ,证明电脉冲可以促使稀有鲫精子摄入外源基因     

Intracellular ciliated protozoal infection in silverlip pearl oysters, Pinctada maxima (Jameson, 1901)

The pathology associated with an intracellular ciliate infection in the digestive gland of pearl oysters Pinctada maxima (Jameson, 1901) is described. Histopathological and transmission electron microscopic examination were used to characterise the organism and its location within host cells. The parasite is tear-drop shaped measuring 5.53 μm (range of 2.73-7.47 μm, n = 9) in width and 11.15 μm (range of 9.02-16.2 μm) in length with a centrally located lobulated nucleus and a large nucleus:cytoplasmic ratio. The ciliate has nine evenly spaced rows of cilia running obliquely along the length of cell, converging on the pointed end. Infected digestive glands typically had a moderate to severe infiltration with mononuclear hemocyte. A strong correlation existed between the burden of ciliates and the host response; (p < 0.001, C = 0.315 Pearson Correlation). The use of a single tissue section upon microscopic examination was found to detect only 38-50% of the infections. However, examination of serial haematoxylin and eosin stained sections improved the reliability of detecting infection.     

Characterization and Quantification of Chitosan Extracted from Nacre of the Abalone Haliotis tuberculata and the Oyster Pinctada maxima

This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan. Received February 16, 2000; accepted July 4, 2000.     

Population genetics of a marine bivalve, Pinctada maxima, throughout the Indo-Australian Archipelago shows differentiation and decreased diversity at range limits

Intraspecific genetic diversity governs the potential of species to prevail in the face of environmental or ecological challenges; therefore, its protection is critical. The Indo-Australian Archipelago (IAA) is a significant reservoir of the world's marine biodiversity and a region of high conservation priority. Yet, despite indications that the IAA may harbour greater intraspecific variation, multiple-locus genetic diversity data are limited. We investigated microsatellite DNA variation in Pinctada maxima populations from the IAA to elucidate potential factors influencing levels of genetic diversity in the region. Results indicate that genetic diversity decreases as the geographical distance away from central Indonesia increases, and that populations located towards the centre of P. maxima 's range are more genetically diverse than those located peripherally ( P <  0.01). Significant partitioning of genetic variation was identified ( F _ST = 0.027; R _ST = 0.023, P  < 0.001) and indicates that historical biogeographical episodes or oceanographic factors have shaped present population genetic structure. We propose that the genetic diversity peak in P. maxima populations may be due to (i) an abundance of suitable habitat within the IAA, meaning larger, more temporally stable populations can be maintained and are less likely to encounter genetic bottlenecks; and/or (ii) the close proximity of biogeographical barriers around central Indonesia results in increased genetic diversity in the region because of admixture of genetically divergent populations. We encourage further genetic diversity studies of IAA marine biota to confirm whether this region has a significant role in maintaining intraspecific diversity, which will greatly assist the planning and efficacy of future conservation efforts.     

以精子为载体的体外转基因猪胚胎研究

去精清精子与外源DNA共孵育,使其携带外源DNA,再与成熟卵母细胞进行体外受精,生产转基因猪胚胎,为通过胚胎移植生产转基因猪奠定基础。结果:通过PCR从不同时期胚胎中检测出携带外源DNA的阳性胚,说明精子与外源DNA共孵育能使精子携带外源DNA,并通过体外受精技术使外源DNA进入早期胚胎。     

Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta)

We investigated gene transfer in abalone via electroporated sperm. The mobility of sperm electroporated either in seawater or in marine invertebrate physiological solution was as good as that of the control group. The fertilization rate reached as high as 94.7--99.6% (93.0-- 99.7% for the control group) when 200 eggs were fertilized by 106 or 107 sperm treated with electroporation at 10 kV and 27 pulses for six cycles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 g and at voltages ranging from 2 to 10 kV, at 27 or 211 pulses for six or 12 cycles showed no differences from the control sperm. After DNase digestion, the genome of the electroporated sperm was analysed by polymerase chain reaction, and it was shown that a 138-bp product was amplified, corresponding to the transgene's amplification product. Southern blotting also showed that a positive band located at the same position as that of opAFP-2000CAT was found in the electroporated sperm after DNase treatment. Analysis by PCR of the genome isolated from a trochophore-stage abalone larva, derived from sperm electroporated with 3.2 g opAFP- 2000CAT, showed the existence of foreign DNA in 13 out of 20 examined samples (65%). The integration of the transferred DNA into the genome of transgenic abalone was also shown by Southern blot analysis. Furthermore, CAT activity was positive for the experimental larvae, but the level of CAT expression was lower than that of larvae derived from sperm electroporated with pCAT- Control vector, driven by SV40 promoter and enhancer sequences. These results demonstrate the potential for the use of sperm as mass gene transfer strategy in marine mollusks such as abalone     

Motility activation and metabolism characteristics of spermatozoa of the black-lip-pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)

Motility of Pinctada margaritifera (Linnaeus, 1758); var: cumingii (Jameson, 1901) (P. margaritifera) spermatozoa collected from gonads are not immediately activated at spawning in seawater (SW) but motility occurs when spermatozoa are transferred into alkaline seawater (pH ranging from 9.0 to 11.4). This motility-activating effect of alkaline pH is reversed when pH is shifted back to more acidic values. In both cases, activity of sperm (% motile cells) increases gradually after alkaline pH activation then lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 5 to 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is from 220 to 230 μm/sec. The general swimming pattern is almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural seawater at 4°C retain potent motility for several days and can be subsequently activated by alkaline seawater. Respiration and ATP concentration were measured in 3 conditions: regular seawater (pH 7.8), artificial diluent (pH 8.2), and alkaline Tris-buffered seawater (pH 10.5). Results show that sperm respiration rates are higher whereas ATP levels are lower in the latter two media.     

猕猴精子优选及体外获能的研究

选择活率高的精子并进行体外获能是开展猕猴体外受精研究的必要程序, 是研究猕猴受精生物学的重要手段。本实验采用上浮法和Percoll 梯度离心法对猕猴精液进行了优选, 并对处理后的精子形态正常率、精子活率、密度及受精率作了比较, 发现二者差异不显著; 用dbcAMP 和咖啡因使精子获能, 发现只有两种获能剂同时存在才能使猕猴精子获能并使卵母细胞受精。结论为: 上浮法和Percoll 法都是有效的精子优选法, 对受精率的影响差异不显著; dbcAMP 和咖啡因在猕猴精子体外获能时缺一不可。     

Characterization of 31 EST-derived microsatellite markers for the pearl oyster Pinctada martensii (Dunker)

We developed and characterized 31 microsatellite markers from expressed sequence tags of Pinctada martensii (Dunker). The number of alleles per locus ranged from 4 to 18 as determined in 44 individuals from a wild population. The expected heterozygosity ranged from 0.4121 to 0.9436, while the observed heterozygosity ranged from 0.4054 to 0.7273. Most of the loci are in Hardy-Weinberg equilibrium. These markers should be useful for population genetics studies, parentage and genome mapping in this species.