In vitro gene transfection using dendritic poly(L-lysine)

Monodispersed dendritic poly(L-lysine)s (DPKs) of several generations were synthesized, and their characteristics as a gene transfection reagent were then investigated. The agarose gel shift and ethidium bromide titration assay proved that the DPKs of the third generation and higher could form a complex with a plasmid DNA, and the degree of compaction of the DNA was increased by the increasing number of the generation. The DPKs of the fifth and sixth generation, which have 64 and 128 amine groups on the surface of the molecule, respectively, showed efficient gene transfection ability into several cultivated cell lines without significant cytotoxity. In addition, the transfection efficiency of the DPK of the sixth generation was not seriously reduced even if serum was added at 50% of the final concentration into the transfection medium. Because we can strictly synthesize various DPK derivatives, which have several types of branch units, terminal cationic groups, and so on, they are expected to be a good object of study regarding the basic information on the detailed mechanism of gene transfection into cells. We also expect to be able to easily construct DPK-based functional gene carriers, e.g., DPKs modified by ligands such as a sugar chain, which can enable advanced gene delivery in vivo.     

磁性纳米颗粒介导基因转染的研究进展

介绍了磁性纳米颗粒介导基因转染的最新研究进展,面临的主要问题以及将来的发展方向。     

NanoSMGT: transfection of exogenous DNA on sex-sorted bovine sperm using nanopolymer

The objective was to introduce exogenous DNA into commercially sex-sorted bovine sperm using nanopolymer for transfection. In the first experiment, the optimal concentration and ratio of linear-to-circular plasmid was determined for NanoSMGT in unsorted sperm. A second experiment was conducted to transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake occurred in a dose-dependent manner (P < 0.05). The optimal amount of DNA was 10 μg/10⁶ cells. The ratios of linear-to-circular plasmid do not influence the uptake by unsorted sperm cells and none of the tested treatments affected sperm motility and viability. Commercially sex-sorted bovine sperm were able to uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted sperm had decreased DNA uptake in comparison to unsorted sperm (P < 0.05). Neither sperm motility nor viability were affected by nanotransfection. In conclusion, nanopolymer efficiently introduced exogenous DNA into commercially sex-sorted bovine sperm; we inferred that these sperm could be used for production of embryos of the desired sex, a technique named NanoSMGT.     

Generation of transgenic mice by transfection of pronuclear embryos using lipid-DNA complexes

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of beta-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.     

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

Background  

Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.     

Identification and characterization of adipose triglyceride lipase (ATGL) gene in birds

Adipose triglyceride lipase (ATGL) is a triglyceride hydrolysis lipase and is generally related to lipid metabolism in animals. The ATGL gene was well studied in mammals, however very less was known in birds that differed significantly with mammals for lipid metabolism. In this study, cloning, mRNA real time and association analysis was performed to characterize the ATGL gene in birds. Results showed that the obtained ATGL gene cDNA of parrot, quail, duck were 1,651 bp (NCBI accession number: GQ221784), 1,557 bp (NCBI accession number: GQ221783) and 1,440 bp each, encoded 481-, 482- and 279-amino acid (AA) peptide, respectively. The parrot ATGL (pATGL) gene was found to predominantly express in breast muscle and leg muscle, and very higher ATGL mRNA level was also found in heart, abdominal fat and subcutaneous fat. The quail ATGL (qATGL) gene was also predominantly expressed in breast muscle and leg muscle, and then to a much lesser degree in heart. The duck ATGL (dATGL) gene was found to predominantly express in subcutaneous fat and abdominal fat, quite higher ATGL mRNA was also found in heart, spleen, breast muscle and leg muscle. Blast analyses indicated the high homology of ATGL and its patatin region, and moreover, and the active serine hydrolase motif (“GASAG” for “GXSXG”) and the glycine rich motif (“GCGFLG” for “GXGXXG”) were completely conservative among 14 species. Association analyses showed that c.950+24C>A, c.950+45C>G, c.950+73G>A, c.950+83C>T and c.950+128delA of chicken ATGL gene (cATGL) were all significantly or highly significantly with cingulated fat width (CFW) (P < 0.05 or P < 0.01), and c.777−26C>A, c.950+45C>G, c.950+73G>A and c.950+118C>T were all significantly or highly significantly with pH value of breast muscle (BMPH) (P < 0.05).     

Biolistic transfection of neuronal cultures using a hand-held gene gun

Biolistic transfection is a technique in which subcellular-sized particles coated with DNA are accelerated to high velocity to propel them into cells. This method is applicable to tissues, cells and organelles, and can be used for both in vitro and in vivo transformations; with the right equipment, it is simple, rapid and efficient. Here we provide a detailed protocol for biolistic transfection of plasmids into cultured human embryonic kidney (HEK) 293 cells and organotypic brain slices using a hand-held gene gun. There are three major steps: (i) coating microcarriers with DNA, (ii) transferring the microcarriers into a cartridge to make a 'bullet', and (iii) firing the DNA-coated microcarriers into cells using a pulse of helium gas. The method can be readily adapted to other cell types and tissues. The protocol can be completed in 1-2 h.     

Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.     

Evolution of sperm structure and energetics in passerine birds

Spermatozoa exhibit considerable interspecific variability in size and shape. Our understanding of the adaptive significance of this diversity, however, remains limited. Determining how variation in sperm structure translates into variation in sperm performance will contribute to our understanding of the evolutionary diversification of sperm form. Here, using data from passerine birds, we test the hypothesis that longer sperm swim faster because they have more available energy. We found that sperm with longer midpieces have higher levels of intracellular adenosine triphosphate (ATP), but that greater energy reserves do not translate into faster-swimming sperm. Additionally, we found that interspecific variation in sperm ATP concentration is not associated with the level of sperm competition faced by males. Finally, using Bayesian methods, we compared the evolutionary trajectories of sperm morphology and ATP content, and show that both traits have undergone directional evolutionary change. However, in contrast to recent suggestions in other taxa, we show that changes in ATP are unlikely to have preceded changes in morphology in passerine sperm. These results suggest that variable selective pressures are likely to have driven the evolution of sperm traits in different taxa, and highlight fundamental biological differences between taxa with internal and external fertilization, as well as those with and without sperm storage.     

Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Deposition gene transfection using bioconjugates of DNA and thermoresponsive cationic homopolymer

A poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics was applied to a vector for use in deposition transfection. PDMAEMA with a molecular weight of 2.5 × 10(5) g mol(-1) was synthesized by photoinduced radical polymerization. Polyplexes approximately 750 nm in size were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA. The polyplexes had a lower critical solution temperature (LCST) of approximately 30 °C. In addition, they exhibited excellent adsorption and durability on a polystyrene surface, as confirmed by a surface chemical compositional analysis. When HeLa cells and primary cells were cultured on a substrate coated with the polyplexes, high transgene expression and cell viability of more than 90% were obtained at low charge ratios (PDMAEMA/plasmid DNA ratio) ranging from 2 to 8. In addition, transgene expression was sustained for over 2 weeks post-transfection whereas decreased expression was observed 5 days post-transfection when the conventional solution-mediated transfection method was used. Thus, high and sustained transgene expression as well as high cell viability can be realized by using small amounts of PDMAEMA as a deposition transfection material.     

Novel thermoresponsive nonviral gene vector: P(NIPAAm-co-NDAPM)-b-PEI with adjustable gene transfection efficiency

A thermoresponsive cationic copolymer, poly( N-isopropylacrylamide- co- N-(3-(dimethylamino)propyl)methacrylamide)- b-polyethyleneimine (P(NIPAAm- co-NDAPM)- b-PEI), was designed and synthesized as a potential nonviral gene vector. The lower critical solution temperature (LCST) of P(NIPAAm- co-NDAPM)- b-PEI in water measured by UV-vis spectroscopy was 38 degrees C. P(NIPAAm- co-NDAPM)- b-PEI as the gene vector was evaluated in terms of cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. P(NIPAAm- co-NDAPM)- b-PEI copolymer exhibited lower cytotoxicity in comparison with 25 kDa PEI. Gel retardation assay study indicated that the copolymer was able to bind DNA completely at N/P ratios higher than 30. At 27 degrees C, the mean particle sizes of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes decreased from 1200 to 570 nm corresponding to the increase in N/P ratios from 10 to 60. When the temperature changed to 37 degrees C, the mean particle sizes of complexes decreased from 850 to 450 nm correspondingly within the same N/P ratio range due to the collapse of thermoresponsive PNIPAAm segments. It was found that the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes was higher than or comparable to that of 25 kDa PEI/DNA complexes at their optimal N/P ratios. Importantly, the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes could be adjusted by altering the transfection and cell culture temperature.     

High transfection efficiency of poly(4-vinylimidazole) as a new gene carrier

Poly(4-vinylimidazole) (P4V) was obtained by free radical polymerization of 4-vinylimidazole (4V) prepared by decarboxylation of urocanic acid. P4V formed a complex with DNA that exhibited higher transfection effiency on Hela cells than polyethylenimine (PEI), through the proton sponge mechanism of the imidazole groups in the side chain of the P4V, and low cell toxicity.     

The risk and intensity of sperm ejection in female birds

The way females utilize the gametes of different males has important consequences for sexual selection, sexual conflict, and intersexual coevolution in natural populations. However, patterns of sperm utilization by females are difficult to demonstrate, and their functional significance remains unclear. Here, we experimentally study sperm ejection in the fowl Gallus gallus domesticus, where females eject preferentially the sperm of socially subordinate males. We study two measures of sperm ejection, (i) the probability that an ejaculate is ejected ("risk") and (ii) the proportion of semen ejected ("intensity"), and show that both measures are strongly nonrandom with respect to characteristics of the ejaculate, the male, and the female. Sperm ejection neutralized on average 80% of an ejaculate, and while larger ejaculates suffered a higher ejection risk, smaller ejaculates suffered more intense ejection. After controlling for ejaculate volume, we found socially subdominant males suffered higher ejection intensity. After controlling for male and ejaculate effects, we found ejection risk increased and intensity declined as females mated with successive males. Collectively, these results reveal that sperm ejection risk and intensity are at least partly actively caused by female behavior and generate independent selective pressures on male and ejaculate phenotypes.     

Distinct evolutionary patterns of morphometric sperm traits in passerine birds

The striking diversity of sperm shape across the animal kingdom is still poorly understood. Postcopulatory sexual selection is an important factor driving the evolution of sperm size and shape. Interestingly, morphometric sperm traits, such as the length of the head, midpiece and flagellum, exhibit a strong positive phenotypic correlation across species. Here we used recently developed comparative methods to investigate how such phenotypic correlations between morphometric sperm traits may evolve. We compare allometric relationships and evolutionary trajectories of three morphometric sperm traits (length of head, midpiece and flagellum) in passerine birds. We show that these traits exhibit strong phenotypic correlations but that allometry varies across families. In addition, the evolutionary trajectories of the midpiece and flagellum are similar while the trajectory for head length differs. We discuss our findings in the light of three scenarios accounting for correlated trait evolution: (i) genetic correlation; (ii) concerted response to selection acting simultaneously on different traits; and (iii) phenotypic correlation between traits driven by mechanistic constraints owing to selection on sperm performance. Our results suggest that concerted response to selection is the most likely explanation for the phenotypic correlation between morphometric sperm traits.