Cytoplasmic Injection Research Articles

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

Transgene delivery via intracellular electroporetic nanoinjection

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

Efficient TALEN-mediated gene knockout in livestock

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors

The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] gene plasmid (50 ng/μL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 μm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.

The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain (ΔN-hH1.4). The ΔN-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that ΔN-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

Murine embryo development following cytoplasmic injection of linear and condensed DNA

In 1985 Brinster et al. [1] observed that linearized DNA injected in the cytoplasm of mouse zygotes underwent spontaneous supercoiling within 24 h. This finding suggests that DNA prefers and functions best in tertiary structure. In an effort to improve the efficiency of transgenesis by cytoplasmic injection, DNA was condensed with MgCl2to form a three dimensional rod-shaped DNA prior to injection in pronuclear stage murine zygotes. The DNA used was enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) and served as a marker for gene integration and protein expression in culture conditions. The condensed CMV-EGFP construct was injected in the cytoplasm at 3 concentrations (100, n = 816; 425, n = 464; and 625 μg/ml, n = 708). For comparison linear CMV-EGFP construct was injected into the pronucleus (5 μg/ml, n = 196) and into the cytoplasm (625 μg/ml, n = 628). In all treatment groups the control and buffer injected embryos developed similarly. Among DNA treatment groups, the highest development of fluorescing embryos was observed in zygotes injected in the cytoplasm with linear CMV-EGFP (625 μg/ml); however, zygotes injected in the cytoplasm with condensed CMV-EGFP (625 μg/ml) had the highest percentage (44%) of fluorescing embryos, the highest percentage (16.7%) of fluorescing morula and blastocysts, and the lowest percentage of fluorescence mosaicism at every stage of embryo development after 4 d in culture; thereby making it the best method for generating transgenic animals.

Machine vision-based localization of nucleic and cytoplasmic injection sites on low-contrast adherent cells

Automated robotic bio-micromanipulation can improve the throughput and efficiency of single-cell experiments. Adherent cells, such as fibroblasts, include a wide range of mammalian cells and are usually very thin with highly irregular morphologies. Automated micromanipulation of these cells is a beneficial yet challenging task, where the machine vision sub-task is addressed in this article. The necessary but neglected problem of localizing injection sites on the nucleus and the cytoplasm is defined and a novel two-stage model-based algorithm is proposed. In Stage I, the gradient information associated with the nucleic regions is extracted and used in a mathematical morphology clustering framework to roughly localize the nucleus. Next, this preliminary segmentation information is used to estimate an ellipsoidal model for the nucleic region, which is then used as an attention window in a k-means clustering-based iterative search algorithm for fine localization of the nucleus and nucleic injection site (NIS). In Stage II, a geometrical model is built on each localized nucleus and employed in a new texture-based region-growing technique called Growing Circles Algorithm to localize the cytoplasmic injection site (CIS). The proposed algorithm has been tested on 405 images containing more than 1,000 NIH/3T3 fibroblast cells, and yielded the precision rates of 0.918, 0.943, and 0.866 for the NIS, CIS, and combined NIS-CIS localizations, respectively.

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.

335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P &lt; 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were &gt;70% and &lt;10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

201 SUPPRESSION OF Suz12 IN BOVINE PREIMPLANTATION EMBRYOS VIA CYTOPLASMIC SMALL INTERFERING RNA INJECTION

The proper removal of gametic epigenetic marks and coordinated reestablishment of the epigenome are critical to mammalian embryonic development. Suz12 is a member of Polycomb repressive complex 2, known to catalyse trimethylation of lysine 27 on histone 3 (H3K27me3; Pasini et al. 2004); Suz12 has also been shown to interact with Suv39h1 for proper trimethylation of H3K9 (de la Cruz et al. 2007). Our objective in this study was to suppress expression of Suz12 via cytoplasmic injection of small interfering RNA (siRNA) targeted at this gene, and to evaluate the effect on embryo development rates. Bovine zygotes were produced in vitro via standard laboratory procedures. Nineteen hours post-fertilization, presumptive zygotes (n = 3979) were divided into 3 treatment groups: noninjected control (CTL), or injected with a fluorescent dextran marker combined with either a nontargeting siRNA (NULL) or an Suz12-targeting siRNA (SUZ). Embryos were cultured in Bovine Evolve (Zenith Biotech, Canada) with 4 mg mL–1 of BSA (Probumin, Millipore, Billerica, MA) and collected at the 4-cell, 8-cell, morula, and blastocyst stages. Ribonucleic acid was isolated with an RNeasy® Mini Kit (Qiagen, Valencia, CA) from 3 replicates of pooled embryos at each stage (4-cell, n = 15; 8-cell, n = 20; morula, n = 10), except for the blastocyst stage [2 samples (n = 10) collected from the CTL and NULL groups, and 1 sample (n = 3) from the SUZ group]. Each RNA sample was reverse-transcribed into cDNA and diluted for use by quantitative real-time PCR. Relative gene expression levels from each sample were calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA), adjusted according to individual PCR efficiencies for each primer pair (R2 &gt; 0.95) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA), to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). Our data indicate that Suz12 expression was suppressed to undetectable levels in SUZ-treated zygotes at all embryo stages analysed. The blastocyst rate of the SUZ group was extremely low (0.88 ± 0.16% SEM) compared with the CTL (19.87 ± 0.36% SEM) and NULL (5.09 ± 0.36% SEM) groups. Morphologically, SUZ morulae appeared fragmented with fewer larger cells than expected, whereas the NULL and CTL morulae seemed to develop normally. We presume the loss of Suz12 expression during this important developmental time is detrimental to embryo morphology and results in a decreased rate of blastocyst formation. Because of this decrease, we were able to collect only 3 SUZ blastocysts from a total of 227 injected. The microinjection procedure also contributed to significant (P &lt; 0.05) decreases in blastocyst rates of the injected groups as compared with the CTL. Future experiments will explore potential alterations in histone methylation, as well as other epigenetic modifiers in bovine preimplantation embryos, to further elucidate the role of the epigenome in early embryonic development.

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.

161 MODULATION OF TELOMERASE ACTIVITY IN BOVINE EMBRYOS USING CYTOPLASMATIC PLASMID INJECTION

Telomeres are repetitive, noncoding sequences at the ends of linear chromosomes that shorten with each cell division. They play an important role in aging and affect the regenerative capacity of cells. The holoenzyme telomerase rebuilds telomeres and is composed of 2 components, i.e. the catalytic protein component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). In mammals, telomerase is active during embryogenesis, in germ cells and a subset of stem and progenitor cells. In the present study, we set out to express the TERC component alone and then in combination with TERT, the human telomerase complex, in bovine embryos. The human telomerase components are highly homologous to bovine telomerase genes. Here, 3 different expression constructs encoding hTERC, hTERT, and a green fluorescent protein (GFP) reporter were co-injected into bovine zygotes cytoplasm, and three groups of 528, 1865, and 110 zygotes were constituted; hTERC/GFP (Group 1), hTERT/hTERC/GFP (Group 2), and GFP alone (Group 3), respectively. GFP fluorescence was used to identify successfully injected embryos. This method has recently been established in our laboratory. Injected and control embryos were cultured for 7 days to the blastocyst stage in vitro and the impact on early embryonic development and the physiological consequences of an ectopic overexpression of telomerase in early bovine embryos were assayed. We obtained 45 blastocysts with green fluorescence in the first, 192 in the second, and 28 in the third group. Embryos with GFP fluorescence were frozen for subsequent PCR analysis and telomerase activity measurement. Some blastocyts were analyzed using quantitative fluoresence in situ hybridization to monitor telomere length. Control groups were analyzed for the endogenous levels of TERC and TERT. Results indicate that endogenous TERC and TERT are up-regulated in morulae and blastocyts. In this study, we show that human TERC and TERT can be expressed in blastocysts by cytoplasmic plasmid injection in bovine zygotes. Statistical analyses were performed using the JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC, USA). The Tukey-Kramer test was applied to compare the group means. The expression of hTERC alone resulted in a significant extension of telomere length of 280 telomere fluorescence units. Expression of both components also resulted in a significant extension of telomere length. In conclusion, TERC component alone is sufficient to elongate telomere length. The activity measurement showed that telomerase activity in the hTERT and hTERC injected group is 1.77 times higher than in the control group. Findings from this study will allow a comprehensive analysis of the functions of TERT and TERC in early embryogenesis. The ectopic expression of telomerase components in bovine embryos could pave new avenues for generating stem cells and for the development of novel regenerative therapies. Funded by DFG.

Cytoplasmic Injection of Circular Plasmids Allows Targeted Expression in Mammalian Embryos

Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter-controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.

Involvement of the dehydroleucodine alpha-methylene-gamma-lactone function in GVBD inhibition inBufo arenarumoocytes

Dehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts of Artemisia douglasiana Besser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ring. It has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2 of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner in Bufo arenarum fully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown. The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation of Bufo arenarum oocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL). The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL. Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed. Our results indicate that the inhibitory effect on meiosis progression of DhL does not depend only on the activity of the alpha-methylenelactone function, as its hydrogenated derivative, 2H-DhL, in which this function has been inactivated, causes similar effects on amphibian oocytes. However, 2H-DhL was less active than DhL as higher doses were required to obtain a significant inhibition. On the other hand, the analysis of the participation of certain mediators in some of the signalling pathways leading to MPF activation suggests that the Myt1 kinase could be a target of these lactones, while cdc25 phosphatase would not be affected. Besides, the PKA inhibition assays indicate that these lactones would act earlier in the signalling pathways.

Microtubule Acetylation Through HDAC6 Inhibition Results in Increased Transfection Efficiency

The success of viral and nonviral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm and reach the nucleus. We, as well as other researchers, have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules with nocodazole was shown to greatly inhibit cytoplasmic plasmid trafficking, it did not abolish it. It has been demonstrated that a pool of stabilized post-translationally acetylated microtubules exists in cells, and that this acetylation may play a role in protein trafficking. In order to determine whether this modification could account for the residual DNA trafficking in nocodazole-treated cells, we inhibited or knocked down the levels of the tubulin deacetylase, histone deacetylase 6 (HDAC6), thereby generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not because of increased transcriptional activity, but rather, because of increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild-type cells. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.

Multiple rescue factors within a Wolbachia strain.

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow "imprints" the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod(+) resc(+), mod(-) resc(+), mod(-) resc(-), and mod(+) resc(-). A so-called "suicide" mod(+) resc(-) strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod(+) resc(-) strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types.

Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

Effect of dehydroleucodine on meiosis reinitiation inBufo arenarumdenuded oocytes

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.

Application of Cloning Technology for Production of Human Polyclonal Antibodies in Cattle

Cloning and Stem CellsVol. 9, No. 1 EssaysApplication of Cloning Technology for Production of Human Polyclonal Antibodies in CattleJames M. RoblJames M. RoblSearch for more papers by this authorPublished Online:26 Mar 2007https://doi.org/10.1089/clo.2006.0083AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byAnimal Health and Welfare10 November 2020Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes16 March 2017 | PLOS ONE, Vol. 12, No. 3Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattleAnimal Reproduction Science, Vol. 136, No. 4Vaccination of Rabbits with an Alkylated Toxoid Rapidly Elicits Potent Neutralizing Antibodies against Botulinum Neurotoxin Serotype BClinical and Vaccine Immunology, Vol. 17, No. 6Year of the ox1 February 2009 | Nature Biotechnology, Vol. 27, No. 2 Volume 9Issue 1Apr 2007 InformationMary Ann Liebert, Inc.To cite this article:James M. Robl.Application of Cloning Technology for Production of Human Polyclonal Antibodies in Cattle.Cloning and Stem Cells.Apr 2007.12-16.http://doi.org/10.1089/clo.2006.0083Published in Volume: 9 Issue 1: March 26, 2007PDF download