Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts

Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350) and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage (P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from 3.2 ± 0.8% to 43.0 ± 3.4% while survival decreased from 90.5 ± 8.0% to 44.8 ± 2.0%. The number of pulses at 300 V significantly affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1–5 pulses from 63.2 ± 6.3% to 3.0 ± 0.3% with GFP expression of surviving cells increasing from 35.6 ± 2.67% to 71.4 ± 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene. These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 μL of 1 × 10⁶ cells/mL in the presence of 12.5 μg DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer resulted in the production of live offspring.     

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.     

Clinical relevance and functional implications for human leucocyte antigen‐g expression in non‐small‐cell lung cancer

HLA‐G has been documented both in establishment of anti‐tumour immune responses and in tumour evasion. To investigate the clinical relevance of HLA‐G in non‐small‐cell lung cancer (NSCLC), expression status and potential significance of HLA‐G in NSCLC were analysed. In this study, HLA‐G expression in 101 NSCLC primary lesions and plasma soluble HLA‐G (sHLA‐G) from 91 patients were analysed with immunohistochemistry and ELISA, respectively. Correlations between HLA‐G status and various clinical parameters including survival time were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression and plasma sHLA‐G form NSCLC patients on natural killer (NK) cell cytolysis were performed. Data revealed that HLA‐G was expressed in 41.6% (42/101) NSCLC primary lesions, while undetectable in adjacent normal lung tissues. HLA‐G expression in NSCLC lesions was strongly correlated to disease stages (P= 0.002). Plasma sHLA‐G from NSCLC patients was markedly higher than that in normal controls (P= 0.004), which was significantly associated with the disease stages (I versus IV, P= 0.025; II versus IV, P= 0.029). Patient plasma sHLA‐G level (≥median, 32.0 U/ml) had a significantly shorter survival time (P= 0.044); however, no similar significance was observed for the lesion HLA‐G expression. In vitro data showed that both cell surface HLA‐G and patient plasma sHLA‐G could dramatically decrease the NK cell cytolysis. Our findings indicated that both lesion HLA‐G expression and plasma sHLA‐G in NSCLC is related to the disease stage and can exert immunosuppression to the NK cell cytolysis, indicating that HLA‐G could be a potential therapeutic target. Moreover, plasma sHLA‐G in NSCLC patients could be used as a prognosis factor for NSCLC.     

Aberrant human leucocyte antigen‐G expression and its clinical relevance in hepatocellular carcinoma

The clinical relevance of human leucocyte antigen‐G (HLA‐G) has been postulated in malignancies. Hepatocellular carcinoma (HCC) is a major contributor to cancer incidence and mortality worldwide; however, potential roles of HLA‐G in HCC remain unknown. In the current study, HLA‐G expression in 219 primary HCC lesions and their adjacent non‐tumourous samples was analysed with immunohistochemistry. Correlations among HLA‐G expression and various clinical parameters were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression on NK cell cytolysis was performed in vitro. HLA‐G expression was observed in 50.2% (110/219) of primary HCC lesions, and undetectable in corresponding adjacent normal liver tissues. HLA‐G expression was found in 37.8%, 41.9% and 71.4% of stage I, II and III HCC lesions, respectively. Data revealed that HLA‐G expression in HCC was strongly correlated to advanced disease stage (I versus II, P= 0.882; I versus III, P= 0.020; II versus III, P= 0.037). HLA‐G expression was also more frequently observed in elder patients (≥median 52 years, 57.5%versus 43.4%, P= 0.004). Meanwhile, plasma soluble HLA‐G in HCC patients was significantly higher than that in normal controls (median, 92.49U/ml versus 9.29U/ml, P= 0.000). Functional assay showed that HLA‐G expression in transfected cells could dramatically decrease the NK cell cytolysis (P= 0.036), which could be markedly restored by the blockade of HLA‐G (P= 0.004) and its receptor ILT2 (P= 0.019). Our finding indicated that HLA‐G expression was strongly correlated to advanced disease stage, and more frequently observed in elder patients. Its relevance to HCC progression might be result from the inhibition of NK cell cytolysis.     

UVB effects on early developmental stages of commercially important macroalgae in southern Chile

High levels of ultraviolet-B radiation (UVB) could represent a danger to seaweeds by affecting their physiological processes and development. The aim of this work was to study the effects of UVB radiation on early developmental stages of commercially and ecologically important marine algal species in southern Chile, considering spores survival and embryos growth. Spores of Mazzaella laminarioides, Gigartina skottsbergii, Sarcothalia crispata and embryos of G. skottsbergii and Macrocystis pyrifera were submitted to treatments of a) photosynthetically active radiation (PAR: Control), b) PAR+UVA (PA) and c) PAR+UVA+UVB (PAB). UV radiation did not affect spore survival of M. laminarioides S. crispata and G. skottsbergii (P = 0.55, P = 0.6 & P = 0.25 respectively), but did provoke differences in the growth rate of G. skottsbergii embryos (P = 0.00). Differences in survival and growth of M. pyrifera embryos were also observed (P = 0.001 & P = 0.007, respectively). Differences in growth of M. pyrifera embryos were observed only in the first five days, whereas changes in survival persisted until the end of the experiment. Additionally, UVB provoked morphological alteration in M. pyrifera embryos, as evidenced by progressive curling. These results suggest that the initial stages of the subtidal algae species G. skottsbergii and M. pyrifera cultivated in laboratory conditions were sensitive to UVA and UVB radiation, and their recruitment and development could be affected as well in natural conditions found in southern South America, where the ozone layer has thinned more than in other parts of the planet.     

Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells

To study self‐renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA‐1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty‐five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (×10⁷/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (×10⁷/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples. Mol. Reprod. Dev. 77: 340–347, 2010. © 2010 Wiley‐Liss, Inc.     

Behavioural response of Kootenai white sturgeon (Acipenser transmontanus,Richardson, 1836) early life stages to gravel,pebble, and rubble substrates: guidelines for rearing substrate size

Guidelines for creating rearing substrate for sturgeon early life stages are needed for restoration programmes creating habitats for spawning and rearing of early life stages. To determine the effects of rock size on motile early life stages, experiments were conducted in artificial streams to observe the behaviour of free embryos and larvae of Kootenai River white sturgeon (Acipenser transmontanus) relative to rock size. Most (≥90%) of the free embryos in replicate test streams with 100% gravel, 100% pebble, or 100% rubble hid under rocks, with few moving downstream. There was no difference in downstream movement of free embryos among rock treatments, therefore all rock types provided cover habitat. Similarly, in rock mixture tests, with a variable percentage of pebble, small rubble, or large rubble in different tanks, even fewer free embryos moved downstream. With increasing age, larvae increasingly used the open bottom and velocity refuges downstream of or alongside rocks of any size while drift feeding. Downstream movement of larvae in both rock regime tests was affected by rock size, with significantly reduced movement relative to increasing abundance of large rock (rubble). However, in all rock mixtures, free embryos (and later, larvae when they stopped dispersing) preferred the smallest rock size available (pebble; P = 0.0001). This suggests a strong innate preference of both life stages for small substrate that is likely related to increased survival. A rock mixture of 10% gravel (16–32 mm diameter) and 30–40% pebble (diameter, 30–60 mm) should provide adequate rearing substrate for free embryos and early‐larvae. The remaining 50–60% should be mixed rubble and boulders for spawning and egg rearing.     

Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells

Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.     

Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat Cells.     

脂质体介导VEGF基因对成骨细胞增殖、合成骨钙素以及细胞周期的相关研究

目的:研究脂质体介导血管内皮生长因子(VEGF)基因对成骨细胞增殖、合成骨钙素以及细胞周期的影响。方法:通过脂质体介导的基因转染方法,将携带外源性VEGF重组pcDNA3-hVEGF质粒导入体外培养的成骨细胞,酶联免疫吸附测定法(ELISA)检测转染后细胞中VEGF浓度变化,以判断转染效果;采用细胞计数法检测转染重组质粒的成骨细胞的增殖活性;流式细胞术检测转染重组质粒的成骨细胞周期的变化;ELISA检测转染重组质粒的成骨细胞骨钙素浓度变化。结果:与对照组相比,转染组成骨细胞中VEGF的浓度显著增加,与对照组间差异具有统计学意义(P0.05);转染重组质粒的成骨细胞的增殖能力较对照组显著增强,差异具有统计学意义(P0.05),与对照组相比,转染重组质粒的成骨细胞周期(G2/M+S)%明显增加,差异具有统计学意义(P0.05);转染重组质粒的成骨细胞合成的骨钙素浓度较对照组显著升高,差异具有统计学意义(P0.05)。结论:脂质体介导成骨细胞增加血管内皮生长因子的水平,可促进成骨细胞增殖,增加成骨细胞骨钙素的浓度,从而提高成骨细胞的功能。     

Transient Transfection of Oligodendrocyte Progenitors by Electroporation

The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10–15% of the surviving cells, and provides approximately 10⁴ surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.     

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.     

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos¹. Different structures such as DNA plasmids encoding genes^2-4, small interfering RNA (siRNA) plasmids⁵, small synthetic RNA oligos⁶, and morpholino antisense oligonucleotides⁷ can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)⁸ and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes⁹ and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos^10-12.     

Production and Properties of α-l-Arabinofuranosidase from Various Strains of Corticium rolfsii

Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed: pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N, survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.     

Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cash-mere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.     

3种无脊椎动物对近江牡蛎Crassostrea ariakensis和熊本牡蛎C. sikamea的捕食研究

捕食是影响牡蛎种群建立和牡蛎礁发育的重要生物因子之一。通过室内受控实验测定了日本蟳（Charybdis japonica）、脉红螺（Rapana venosa）和黄口荔枝螺（Thais luteostoma）对4组规格（W1：壳高10-20mm;W2：壳高20-30mm;W3：壳高30-40mm;W4：壳高>40mm）近江牡蛎（Crassostrea ariakensis）和熊本牡蛎（C.sikamea）的捕食偏好性和捕食效率。双因子方差分析结果表明,日本蟳对2种牡蛎的捕食效率没有显著性差异（P>0.05）,但牡蛎规格大小显著影响着日本蟳的捕食效率（P<0.05）,即日本蟳对W1组近江牡蛎的捕食效率显著高于W2和W4组（P<0.05）,W3组的被捕食效率介于中间（P>0.05）;日本蟳对W1组熊本牡蛎的捕食效率显著高于W2和W3组（P<0.05）,W4组的被捕食效率与其他处理组均没有显著性差异（P>0.05）。牡蛎种类（P=0.590）和规格大小（P=0.357）对脉红螺的捕食效率均无显著性影响,不同规格的两种牡蛎均呈现较低的被捕食效率。黄口荔枝螺对2种牡蛎的捕食效率无显著性差异（P=0.917）,但牡蛎规格大小显著影响其捕食效率（P=0.035）,即对W1组熊本牡蛎捕食效率显著高于其他3个规格组（P<0.05）,但其对不同规格近江牡蛎的捕食效率没有显著性差异（P>0.05）。2种牡蛎的壳厚与其壳高之间均存在极显著的正相关关系（P<0.001）。研究结果表明,3种无脊椎动物捕食者对近江牡蛎和熊本牡蛎并未表现出差异性的捕食偏好,但对不同规格牡蛎的捕食效率具有种间差异。     

电穿孔介导小干扰RNA高效转染小鼠附植前胚胎

使用Cy3标记的阴性对照小干扰RNA(siRNA)转染小鼠附植前胚胎,建立向小鼠附植前胚胎导入siRNA的电穿孔方法。通过控制透明带弱化程度、电压、脉冲时间和脉冲次数等条件,采用不同参数组合并结合使用不同介质作为电转缓冲液将Cy3标记的阴性对照siRNA转染小鼠附植前胚胎。在荧光倒置显微镜下,观察胚胎的存活率、siRNA转染率以及阳性转染存活胚胎的囊胚发育率。结果显示小鼠附植前胚胎在使用台氏液消化胚胎透明带10 s后,以opti-MEM作为电转缓冲液,电穿孔参数设置为30 V,1 ms,3次的条件下取得最佳转染效果。总之,电穿孔方法可实现siRNA简便、高效地转染小鼠附植前胚胎。