<Emphasis Type="Italic">Tβ4</Emphasis>-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics

Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. To obtain genetically modified cells as nuclear donors, we co-transfected donor vectors into fetal fibroblasts of cashmere goats. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Via determination of the copy numbers and integration sites, the Tβ4 gene was successfully inserted into the goat genome. Histological examination of skin tissue revealed that Tβ4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tβ4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). Our results indicate that Tβ4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.     

Production of functional human CuZn-SOD and EC-SOD in bitransgenic cloned goat milk

Human copper/zinc superoxide dismutase (CuZn-SOD) and extracellular superoxide dismutase (EC-SOD) are two superoxide dismutases that scavenge reactive oxygen species (ROS). Their biological role of eliminating oxidative stress caused by excessive ROS levels in living organisms has been utilized in medical treatment, preventing skin photoaging and food preservation. In this study, we employed two sequences that encode human CuZn-SOD and EC-SOD, along with goat beta-casein 5′ and 3′ regulatory elements, to construct mammary gland-specific expression vectors. Bitransgenic goats were generated using somatic cell nuclear transfer (SCNT), which employed co-transfection to generate bitransgenic goat fetal fibroblast cells as donor cells, and the expression of human CuZn-SOD and EC-SOD and their biological activities were assayed in the milk. PCR and Southern blot analysis confirmed that the cloned goat harbors both hCuZn-SOD and hEC-SOD transgenes. rhCuZn-SOD and rhEC-SOD were expressed in the mammary glands of bitransgenic goat, as determined by western blotting. The expression levels were 100.14?±?5.09 mg/L for rhCuZn-SOD and 279.10?±?5.38 mg/L for rhEC-SOD, as determined using ELISA. A total superoxide dismutase assay with WST-8 indicates that the biological activity of rhCuZn-SOD and rhEC-SOD in goat milk is 1451?±?136 U/mL. The results indicate that two expression vectors can simultaneously transfect goat fetal fibroblast cells as donor cells to produce transgenic goats by SCNT, and the CuZn-SOD and EC-SOD proteins secreted in the mammary glands showed biological activity. The present study thus describes an initial step in the production of recombinant human SODs that may potentially be used for therapeutic purposes.     

Production of heterozygous alpha 1,3-galactosyltransferase (<Emphasis Type="Italic">GGTA1</Emphasis>) knock-out transgenic miniature pigs expressing human <Emphasis Type="Italic">CD39</Emphasis>

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig’s cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig’s cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.     

Comparative muscle proteomics/phosphoproteomics analysis provides new insight for the biosafety evaluation of <Emphasis Type="Italic">fat</Emphasis>-<Emphasis Type="Italic">1</Emphasis> transgenic cattle

The biosafety of fat-1 transgenic cattle has been a focus of our studies since the first fat-1 transgenic cow was born. In this study, we used tandem mass tag labeling, TiO₂ enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC–MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 transgenic cattle.     

The effect of regulatory sequences of αSl-casein gene on the expression of the <Emphasis Type="Italic">lacZ</Emphasis>-gene in loach <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> L. transgenic embryos

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine αSl-casein gene tissue-specific promoter-enhancer region or highly homologous goat αSl-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1–3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the αSl-casein gene of goat was higher than with the promoter-enhancer region of the bovine αSl-casein gene. Thus, regulatory sequences of the bovine or goat αSl-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Phage integrases for the construction and manipulation of transgenic mammals

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the φC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the φC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.     

SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo

Background

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development.

Results

SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses.

Conclusions

Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

     

Novel SNPs of the bovine <Emphasis Type="Italic">NUCB2</Emphasis> gene and their association with growth traits in three native Chinese cattle breeds

In this study, polymorphism in the exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 of bovine NUCB2 gene was detected by PCR-SSCP and DNA sequencing methods in 686 individuals from three Chinese cattle breeds. Two haplotypes (M and N), three observed genotypes (MM, MN and NN) and two SNPs (NC_007313: g. 27451G>A, NC_007313: g. 27472T>C) were detected. The frequencies of haplotypes M and N in inland Chinese three breeds were 0.531–0.721 and 0.279–0.469 respectively. The studied showed that Nanyang, Jiaxian Red and Qinchuan cattle populations were in Hardy–Weinberg equilibrium at SNPs locus of NUCB2 gene (P > 0.05). Polymorphism of the NUCB2 gene was shown to be associated with growth traits in Qingchuan and Nanyang cattle breed. The linkage of two mutant sites in the bovine NUCB2 gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months (P < 0.05). Results of this study suggested that the NUCB2-gene-specific SNP may be a useful marker for growth traits in future marker-assisted selection programmes in inland Chinese cattle.     

Associations between polymorphisms in the NICD domain of bovine <Emphasis Type="Italic">NOTCH1</Emphasis> gene and growth traits in Chinese Qinchuan cattle

NOTCH1 is one of the four mammalian Notch receptors, which is involved in the Notch signaling pathway. Specifically, NOTCH1 promotes the proliferation of myogenic precursor cells, and the NICD domain of NOTCH1 can impair regeneration of skeletal muscles. However, similar research on the bovine NOTCH1 gene is lacking. In this study, we detected the polymorphisms of the bovine NOTCH1 gene in a total of 448 individuals from Chinese Qinchuan cattle with DNA pooling, forced PCR-RFLP, and DNA sequencing methods. Five novel SNPs were identified within the NICD domain, and eight haplotypes comprising combinations of these five SNPs were studied as well. The association analysis of SNPs’ effects with growth traits revealed that g.A48250G was significantly associated with body height, body weight, and height at hip cross, and that g.A49239C only showed significant associations with body height. This suggests that the NOTCH1 gene is a strong candidate gene that could be utilized as a promising marker in beef cattle breeding programs.     

VPA selectively regulates pluripotency gene expression on donor cell and improve SCNT embryo development

SCNT technology has been successfully used to clone a variety of mammals, but the cloning efficiency is very low. This low efficiency is likely due to the incomplete reprogramming of SCNT embryos. Histone modification and DNA methylation may participate in these events. Thus, it would be interesting to attempt to improve the efficiency of SCNT by using a HDACi VPA. In order to guarantee the effect of VPA and reduce its cytotoxicity, a comprehensive analysis of the cell proliferation and histone modification was performed. The results showed that 0.5 and 1 mM VPA treatment for 24 h were the optimal condition. According to the results, H3K4me3 was increased in 0.5 and 1 mM VPA groups, whereas H3K9me2 was significantly decreased. These are the signals of gene-activation. In addition, VPA treatment led to the overexpression of Oct4 and Nanog. These indicated that VPA-treated cells had similar patterns of histone to zygotic embryos, and may be more favorable for reprograming. A total of 833 cloned embryos were produced from the experimental replicates of VPA-treated donor cells. In 1 mM treatment group, the blastocyst rates were significantly increased compared with control. At the same time, our findings demonstrated the interrelation between DNA methylation and histone modifications.     

Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis

Somatic embryo (SE) regeneration is an ideal experimental system to realize rapid propagation of excellent clones and genetic improvement for perennial gymnosperms. In the present study, genes encoding the miRNA166 precursor were identified and LamiR166a was successfully transformed into the gymnosperm Larix leptolepis (L. leptolepis) and five LamiR166a over-expressed embryonic cell lines were screened out as stable embryo masses. As expected, the targets of miR166a, LaHDZ31-34, were all down-regulated in transgenic lines according to qRT-PCR results. The results showed that the percentage of normal SEs with 4–7 cotyledons was 77.0?% in wild type (WT) lines, but was reduced to 60.3?% in the pSuper::MIR166a lines with “cup-shaped” embryos comprised 7.0?% of WT and 20.7?% of transgenic embryos. Microscopic observation further showed that the intermediate region surrounded by the cotyledons was larger than in the control, with no upward bulge of the shoot apical meristem (SAM). The expression pattern of the two meristem marker genes CLAVATA (CLV) and WUSCHEL-related homeobox (WOX) were investigated. The results showed that the expression levels of WOX were three times higher in transgenic lines than in WT samples, which suggest that miR166a may indirectly regulate SAM development by directly affecting WOX expression. Besides, overexpression of LamiR166a clearly increased the rooting rate and promoted lateral root formation in L. leptolepis seedlings. These results may provide new insights into the regulatory role of miR166 in gymnosperms, and also new applications for forestry production in practice.     

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos

Objectives

We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos.

Results

There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels.

Conclusion

Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

     

Ectopic expression of reprogramming factors enhances the development of cloned porcine embryos

Inefficient cloning by somatic cell nuclear transfer (SCNT) is largely attributed to defects in epigenetic reprogramming. Reprogramming factors (RFs) (Oct4, Sox2, Klf4, c-Myc, Lin28 and Nanog; OSKMLN) can achieve epigenetic reprogramming, suggesting that these might facilitate reprogramming of oocytes. Here, porcine mesenchymal stem cells (pMSCs) treated with exogenous OSKMLN or OSKM were selected as nuclei donors for SCNT. The resulting embryos displayed significantly better development than controls in terms of cleavage rates and blastomere numbers. OSKM treatment improved pluripotency status and regulation of epigenetic factors in modified pMSCs. These changed gene patterns promoted H3K9Ac both in modified pMSCs and their SCNT-derived embryos. Thus, higher histone acetylation levels in donor cells might favor subsequent clone development. Application of exogenous RFs in SCNT offers a novel way for improving cloning efficiency.     

Olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) plants transgenic for tobacco osmotin gene are less sensitive to in vitro-induced drought stress

Olive is one of the most important tree crops in the Mediterranean region, because of its ability to grow and produce acceptable yields under limited water availability. In this study, the drought tolerance of an olive cultivar Canino was compared to the performance of its derived transgenic line expressing osmotin gene from tobacco, obtained by Agrobacterium-mediated transformation of Canino cultivar. Shoot cultures of both wild-type (wt) and transgenic lines were exposed to drought stress over a 28-day period, and their differential responses to in vitro-drought stress were investigated. After exposure to PEG, most of the shoots from wt plants resulted in damage and exhibited decreased levels of chlorophyll, while those of transgenic line did not show injuries and showed a normal growth even when exposed to the highest PEG concentration (4%). After preliminary evaluation we characterized Canino AT17-1, by measuring several physiological parameters, including the activities of the antioxidant enzymes (POD and CAT), and the content of malondialdehyde (MDA). Both the activity of catalase and the proline content were higher in the leaves of the transgenic shoots compared to wt plants. Consequently, it was observed that the transgenic line accumulated less MDA indicating that the presence of the osmotin gene protected the cell membrane from damage by lipid peroxidation. Together, these results could suggest that the transgenic line Canino AT17-1 was more efficient in the activation of defense responses against oxidative stress with respect to the Canino wt. The further finding that the transgenic shoots also showed higher proline accumulation supported the hypothesis that the osmotin gene conferred to transgenic shoots increased tolerance to drought stress compared with the wt.     

Biofilm production and other virulence factors in <Emphasis Type="Italic">Streptococcus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> isolated from clinical cases of bovine mastitis in Poland

Background

Mastitis is a common disease in dairy cattle throughout the world and causes considerable economic losses each year. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among bacteria of the genus Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland.

Results

Most of the isolates analysed demonstrated an ability to produce biofilm (over 70%). Virulence genes were searched for in the three most common streptococci in our experiment: S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33%), cylE (78%), bca (37%), and cfb (100%). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes of the species were Ia (n?=?8) and II (n?=?8), in addition to some strains that were not classified in any of the groups (n?=?6). Out of the eight selected genes for S. uberis (sua, pauA/skc, gapC, cfu, lbp, hasA, hasB, hasC), only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76% and 86% of the strains, respectively.

Conclusions

The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.