显微注射

To obtain the fertilized eggs with clear male pronuclei is the key of success .

显微注射法是制备转基因动物的首选方法，而原核清晰受精卵的获得是影响显微注射成败的关键。

Mathematical model of microinjection amount is founded through depth theory research to the microinjector.

本文通过对显微注射仪的深入研究，建立了注射量的数学模型。

Briefly, a DNA construct or PCR product which carried gene of interest is mixed with a co-injection marker and injected intothe distal gonad through a micropipette.

显微注射技术使用尖端开口直径为微米级的玻璃微管，将所研究的目的DNA注射进线虫的性腺。

Briefly, a DNA construct or PCR product which carried gene of interest is mixed with a co-injection marker and injected into the distal gonad through a micropipette.

显微注射技术使用尖端开口直径为微米级的玻璃微管，将所研究的目的DNA注射进线虫的性腺。

To make the transgenic mice: We purified the gene fraction nestin-hsp68-EGFP and injected it into pronucleus of mouse zygotes to generate the transgenic mice.

1。转基因小鼠的获得：纯化基因片段nestin-hsp68-EGFP，用显微注射方法把基因片段注射到受精卵的雄原核中，获取转基因小鼠。

Recombinant expression vector k14/c-myc-paav was constructed bearing the mouse c-Myc cDNA derived by the skin specific human keratin 14 promoter, then the linear vector was microinjected into the two pronucleus of the C57BL/6 fertilized eggs to produce transgenic mice.256 microinjected eggs were transferred, 6 of the foster mothers were pregnant and 44 adult mice were produced, two of them were positive transgenic mice confirmed by PCR and Southern blot analysis, transgenic rate was about 4%.

以CMV-PAAV载体为基础载体，以皮肤特异人K14（人角蛋白14）基因启动子替换原质粒中的CMV启动子后，在其多克隆位点区依次插入鼠c-Myc基因的cDNA序列和gfp基因的编码序列，构建了高效表达c-Myc基因的真核表达载体。通过双原核显微注射技术，将线性化的载体DNA注入到C57BL/6小鼠受精卵的两个原核中，然后将注射后状态良好的胚胎移植到代孕鼠的输卵管中，共移植了256枚受精卵，6只受体怀孕，最终获得44只成年小鼠。

The healthy survivals were also transferred into the oviducts of pseudopregnant female mice. PCR was used to analyze the integration of the transgene in the genomes of mice.

选用内交系C57BL／6J种小鼠作为受精卵的供体鼠，昆明种小鼠作为假孕母鼠，将长2583bp、携带有CMV启动子和人CKLF-1基因的片段显微注射到受精卵的原核内，然后将注射后成活且健康的受精卵移植到假孕母鼠的输卵管内使其发育。

MATERIALS AND METHODS: Arg to Ser mutation was introduced into the 249 position of the p53 gene by knock-in method. These ES cells with this mutation were selected according to the homologues-recombination with PCR and Southern blot. The positive ES cells without a selection marker were injected into blastocysts recovered from Hprt(superscript -/-) mice, which were derived from Hprt-deficient ES cells. The injected blastocysts then were implanted into pseudopregnant females.

材料与方法：利用基因打靶技术在小鼠胚胎干细胞p53基因249编码子中引入点突变，使编码子249由精氨酸变成丝氨酸，然后将含突变的ES细胞显微注射到Hprt小鼠囊胚中，将注射过的囊胚植入假孕的雌性小鼠子宫，到第14d取小鼠胚胎纤维母细胞，用含HAT的培养液筛选出从ES细胞分化而成的鼠EF细胞，经测序证实细胞含有由249Arg到Ser的突变。

Six hundred ova were transfered into twenty-nine pseudopregnant recipients. Fifty-five offspring were born from the ova mircroinjected with the Bcl-2 fragment expression cassette.Twelve of them were proved to be gene integration positive by PCR .Four founder mice,two male and two female ,were further confirmed to be transgenic by Southern hybridization.

用BglII+SalI+PvuI三酶切pWBS和用BglII+BamHI双酶切pWCS，分别回收并纯化3.0kb的基因片段WAP-Bcl-2-SV40polyA和3.5kb的基因片段WAP-c-myc-SV40poly，作为目的基因，通过受精卵显微注射技术，分别导入C57BL／6J雌鼠与DBA／2J雄鼠交配的F1代小鼠受精卵的雄原核中，各注射约1000枚，将存活的受精卵各600枚分别移植至29只假孕母鼠输卵管壶腹部内。

Blastocyst rate of embryos co-cultured with pig oviduct epithelium cells came from pig oviduct during diestrus was significantly lower than metestrous. 4. The blastocyst rate of transgenic embryos collected at 24~26 hours and 18~22 hours after hCG-treated reached 45% and 20% respectively.

实验证明在hCG注射后24~26h之间获得的受精卵，在原核显微注射人血清白蛋白基因后体外培养，有45%注射胚发育到囊胚阶段，而在18~22h仅有20%，二者差异显著（P.05）。5。

On the other hand, the more important thing is because the urban housing is a kind of heterogeneity products.

另一方面，更重要的是由于城市住房是一种异质性产品。

Climate histogram is the fall that collects place measure calm value, cent serves as cross axle for a few equal interval, the area that the frequency that the value appears according to place is accumulated and becomes will be determined inside each interval, discharge the graph that rise with post, also be called histogram.

气候直方图是将所收集的降水量测定值，分为几个相等的区间作为横轴，并将各区间内所测定值依所出现的次数累积而成的面积，用柱子排起来的图形，也叫做柱状图。