Asymmetric nuclear reprogramming in somatic cell nuclear transfer?

Despite the progress achieved over the last decade after the birth of the first cloned mammal, the efficiency of reproductive cloning remains invariably low. However, research aiming at the use of nuclear transfer for the production of patient-tailored stem cells for cell/tissue therapy is progressing rapidly. Yet, reproductive cloning has many potential implications for animal breeding, transgenic research and the conservation of endangered species. In this article we suggest that the changes in the epi-/genotype observed in cloned embryos arise from unbalanced nuclear reprogramming between parental chromosomes. It is probable that the oocyte reprogramming machinery, devised for resident chromosomes, cannot target the paternal alleles of somatic cells. We, therefore, suggest that a reasonable approach to balance this asymmetry in nuclear reprogramming might involve the transient expression in donor cells of chromatin remodelling proteins, which are physiologically expressed during spermatogenesis, in order to induce a male-specific chromatin organisation in the somatic cells before nuclear transfer.     

Cloning animals by somatic cell nuclear transfer – biological factors

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle.     

Handmade cloning: the future way of nuclear transfer?

The topic of this review is an alternative technique for somatic cell nuclear transfer. Removal of the zona pellucida facilitates manipulations of mammalian oocytes and early embryos, and problems related to their subsequent culture are commonly overestimated. This approach enables radical modifications to somatic cell nuclear transfer, and the handmade cloning (HMC) technique is now successfully applied to an increasing numbers of species. HMC radically decreases costs and the need for a skilled workforce; furthermore, it increases productivity, enables cryopreservation, and results in birth rates comparable, or even higher, than those achievable by micromanipulation-based traditional cloning (TC). The new technique can accelerate technology transfer and standardization and, eventually, might contribute to the widespread application of cloning. Additionally, HMC offers unique possibilities for the automation of somatic cell nuclear transfer.     

Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming?

Fertile offspring from somatic cell nuclear transfer (SCNT) is the goal of most cloning laboratories. For this process to be successful, a number of events must occur correctly. First the donor nucleus must be in a state that is amenable to remodeling and subsequent genomic reprogramming. The nucleus must be introduced into an oocyte cytoplasm that is capable of facilitating the nuclear remodeling. The oocyte must then be adequately stimulated to initiate development. Finally the resulting embryo must be cultured in an environment that is compatible with the development of that particular embryo. Much has been learned about the incredible changes that occur to a nucleus after it is placed in the cytoplasm of an oocyte. While we think that we are gaining an understanding of the reorganization that occurs to proteins in the donor nucleus, the process of cloning is still very inefficient. Below we will introduce the procedures for SCNT, discuss nuclear remodeling and reprogramming, and review techniques that may improve reprogramming. Finally we will briefly touch on other aspects of SCNT that may improve the development of cloned embryos.     

Ultrasonographic fetal parameters and neonatal survival in somatic cell nuclear transfer–derived beef calves

The objectives of this study were to identify prognostic indicators of calf survival in SCNT-derived beef calves. Ultrasonographic parameters of fetal well-being and development, maternal clinical parameters, and neonatal parameters were evaluated as predictors of calf survival in cows carrying SCNT-derived beef fetuses (n = 38). Calf survival was 61.5% (88.2% female and 40.9% male calves; P = 0.0026). Cow respiratory rate and cow temperature were significantly greater in the nonsurviving (NS) group 1 week prepartum. In surviving (S) calves, fetal heart rate (FHR) decreased during the last 2 weeks of gestation (P < 0.01). However, this final deceleration was not observed in NS calves, resulting in higher FHRs in this group (P < 0.0001). Fetal movement and fluid scores did not differ with calf classification. Mean amniotic fluid depth was smaller in S (5.5 ± 0.7 cm) than NS (8.7 ± 1.4 cm) calves (P = 0.0398). However, mean allantoic fluid depth did not differ (P = 0.6120). There was a significant association between the body weight of calf and the diameter of the fetal aorta (P = 0.0115; r² = 0.3762). Surviving calves were lighter at birth (P = 0.0028) and were born later (P = 0.007) than NS calves. Calves born vaginally had a smaller fetal aorta (2.1 ± 0.1 cm vaginal and 2.4 ± 0.1 cm Cesarean) (P = 0.0487) and a lighter birth weight (41.4 ± 4.2 kg vaginal and 60.4 ± 2.1 kg Cesarean) (P = 0.0001) than calves born by Cesarean. Also, calves that underwent spontaneous labor (52.2% S and 0% NS; P = 0.0029) had a lighter birth weight (44.9 ± 3.8 kg) than calves that did not initiate labor (61.6 ± 2.2 kg) (P = 0.0004). Frequent ultrasonographic fetal monitoring allowed identification of differences between S and NS calves. Calves without a final decrease in FHR or with a large aortic diameter were more likely to require a Cesarean because of failure to initiate labor or fetomaternal disproportion. Parameters of fetal well-being and development during the last 3 weeks of gestation were first described in SCNT-derived beef calves.     

Generation of goats by nuclear transfer: a retrospective analysis of a commercial operation (1998–2010)

At LFB USA, Inc., the ultimate use for transgenic cloned goats is for the production of recombinant human protein therapeutics in their milk. This retrospective analysis of the Somatic Cell Nuclear Transfer (SCNT) program, spanning from 1998 to 2010, examined parameters potentially affecting the outcomes and efficiencies in this commercial operation. Over 37,000?+?ova were utilized in the SCNT protocol producing a total of 203 cloned goats. Fifty one (51) clones were produced from non-transfected (transgenic and non-transgenic animal donor) cell lines and 152 clones were produced from transfected cell lines. Comparisons and summaries of (a) transfected versus non-transfected cell lines, (b) relationship of SCNT parameters to offspring produced, (c) skin versus fetal cells, (d) fresh versus cryopreserved cells, (e) parameters from all cell lines used versus those producing SCNT offspring, (f) variation among cell sources, (g) methods of SCNT parturition management and effects on live offspring, and lastly (h) SCNT variation by program are reported. Findings indicate that (a) non-transfected cell lines were more efficient versus transfected cell lines in generating viable cloned offspring on a per reconstructed embryo transferred basis, (b) transfected fetal fibroblasts had improved efficiency versus transfected skin fibroblasts, (c) the percentage of non-transfected cell lines that produced offspring was statistically higher than transfected cell lines, (d) and induction of parturition improved the percentage of viable offspring. In summary, this retrospective analysis on the SCNT process has identified certain parameters for improved efficiency in producing viable cloned goats in a commercial setting.

     

Can nuclear localization signals enhance nuclear localization of plasmid DNA?

Nonviral vectors are safer and more cost-effective than viral vectors but are significantly less efficient, and thus, increasing the efficiency of nonviral vectors remains an important objective. One way to overcome this problem is by stimulating the nuclear localization of exogenous genes. Nuclear localization signals (NLSs) are known to be involved in the active transport of exogenous proteins and probes into the nucleus. However, stimulation of nuclear localization of plasmid DNA has yet to be confirmed completely. In the present study, we prepared plasmid DNA-NLS peptide conjugates and adjusted spacer length and number introduced in an attempt to increase transfection efficiency. In comparison to conjugates with unmodified plasmid DNA and short spacers, we found that NLS-plasmid DNA conjugates with covalent bonding by diazo coupling through PEG chain (MW 3400) stimulated complexation with the nuclear transport proteins importin alpha and importin beta. Evaluation of transfection showed higher expression efficiency with plasmid DNA-NLS peptide conjugates than with unmodified plasmids. However, evaluation of intracellular trafficking after microinjection into the cytoplasm showed plasmid DNA-NLS peptide conjugates only within the cytoplasm; there was no NLS-plasmid stimulation of nuclear localization. Our findings suggest that stimulation of plasmid nuclear localization cannot be achieved merely by changing spacer length or chemically modifying plasmid DNA-NLS peptide conjugates. An additional mechanism must be involved.     

Defects in trophoblast cell lineage account for the impaired in vivo development of cloned embryos generated by somatic nuclear transfer

The low success rate of somatic nuclear transfer (NT) is hypothesized to be mainly due to functional defects in the trophoblast cell lineage rather than the inner cell mass (ICM); this hypothesis, however, remains to be tested directly. Here we separated the ICMs from cloned blastocysts and aggregated the cloned ICM with two fertilization-derived (FD) tetraploid (4N) embryos. We found that the full-term development of cloned ICMs was dramatically improved after the trophoblast cells in the cloned blastocysts were replaced by cells from tetraploid embryos, thus providing direct evidence that defects in trophoblast cell lineage underlie the low success rate of somatic NT.     

A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

1. A system has been developed for the specific transfer of [(3)H]dihydrotestosterone-receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [(3)H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [(3)H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.     

Large offspring or large placenta syndrome? Morphometric analysis of late gestation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois

Somatic nuclear transfer (NT) in cattle is often complicated by fetal oversize (i.e., large offspring syndrome), hydrallantois, and placentomegaly in late gestation. The aims of this work were to obtain data on the placentome structure in NT-recipient cows with hydrallantois (NTH) and to relate these with fetal and placental weights to better understand the abnormalities observed in NTH pregnancies during the third trimester. Pregnant cows were slaughtered between Gestation Days 180 and 280. The fetuses were weighed, and the placentomes were numbered and weighed. Placentomes were examined by histologic and stereological techniques. Macroscopic data showed that placental overgrowth preceded fetal overgrowth, and the ratio of the fetal to the total placentome weight in the NTH group was lower than that in controls after Gestation Day 220. This suggests that placental overgrowth is due to placental default rather than due to fetal overgrowth, as shown also by stereological analysis showing primary deregulation of the growth of cotyledonary tissues. Observed alterations, such as thinning of the maternal epithelium within placentomes and increased trophoblastic surface, could be secondary adaptations. Thus, placental growth deregulations would be due to modifications of the expression of placental factors. Various examples of placental deficiency were observed, suggesting that some fetal abnormalities observed in NTH calves, such as enlarged heart, enlarged umbilical cord, and abdominal ascites, are consequences of placental dysfunction. Therefore, the condition described by the term "large offspring syndrome" might better be described by "large placenta syndrome," because this syndrome affects an average of 50% of late-gestation NT pregnancies. No conclusion can be drawn from this work on apparently normal pregnancies.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.     

Förster-type energy transfer. Simultaneous 'forward' and 'reverse' transfer between unlike fluorophores

The general case of F?rster-type energy transfer is that in which energy is exchanged in both directions between two unlike fluorophores. In such cases, energy is transferred from the conventionally defined donor to the conventionally defined acceptor (forward transfer) and at the same time from the acceptor to the donor (reverse transfer). Expressions are derived to describe the fluorescence intensities and lifetimes of fluorophores undergoing simultaneous forward and reverse transfer; these are compared with corresponding quantities for the case more usually considered, in which only forward transfer is significant. It is shown that the presence of reverse transfer removes the distinction between donor and acceptor, and allows such anomalous effects as 'acceptor quenching'. A confirmatory example is described. It is shown that the equations generally used in distance determination by steady-state fluorescence spectroscopy can also be applied in the presence of reverse transfer, if a correction term is included; however, for lifetime spectroscopy the correction is more complex.     

Attempts to observe through-chain energy transfer and electron transfer on α-helical polypeptide chain

Singlet singlet energy transfer between the two terminal chromophores attached to an α-helical polypeptide chain has been studied. The transfer efficiency was satisfactorily explained by Förster's theory when the interchromophore distance was calculated from the α-helical structure. Therefore, it was concluded that no particular effect from the possible energy band structure of the α-helical conformation was detected in the end-to-end energy transfer. Similarly, end-to-end electron transfer was attempted between the electron donor acceptor pair attached to the ends of α-helcial polypeptide chain. However, no intramolecular interaction was found between the donor acceptor pair, indicating that the exciton structure of the α-helical polypeptides is not effective enough to realize through-chain electron transfer.     

RNA surveillance by nuclear scanning?

There are many quality-control mechanisms that ensure high fidelity of gene expression. One of these is the nonsense-mediated decay (NMD) pathway, which destroys aberrant mRNAs that contain premature termination codons generated as a result of biosynthetic errors or random and programmed gene mutations. Two complexes that initially bind to RNA in the nucleus have been suggested to be involved in NMD in the cytoplasm. Here we propose an alternative model that involves nuclear scanning, on the basis of recent evidence for nuclear translation.