The changing role of cell culture in the generation of transgenic livestock

Transgenesis may allow the generation of farm animals with altered phenotype, animal models for research and animal bioreactors. Although such animals have been produced, the time and expense involved in generating transgenic livestock and then evaluating the transgene expression pattern is very restrictive. If questions about the ability and efficiency of expression could be asked solely in vitro rapid progress could be achieved. Unfortunately, experiments addressing transcriptional control in vitro have proved unreliable in their ability to indicate whether a transgene will be transcribed or not. However, initial studies suggest that cell culture may be able to predict in vivo post-transcriptional events. We review these issues and propose that strategies which engineer the transgene integration site could enhance the probability for efficient expression. This approach has now become feasible with the development of techniques allowing animals to be generated from somatic cells by nuclear transfer. The important step in this procedure is the use of cells grown in culture as the source of genetic information, allowing the selection of specific transgene integration events. This technology which has dramatically increased the potential use of transgenic livestock for both agricultural and biotechnological applications, is based on standard cell culture methodology. We are now at the start of a new era in large animal transgenics

Pertumbuhan Bakteri Laut Shewanella Indica LBF-1-0076 Dalam Naftalena Dan Deteksi Gen Naftalena Dioksigenase - (the Growth of Marine Bacteria Shewanella Indica LBF-1-0076 in Naphthalene and Naphthalene Dioxygenase Gene Detection)

Crude oil exploitation which often occured offshore can cause water pollution in the sea since its contains naphthalene which is a hazardous compounds. This research used marine bacteria LBF-1-0076 that have ability in naphthalene degradation. This research aimed to study the parameter effect of naphthalene and cell concentration toward marine bacteria LBF-1-0076. This research also identified isolate LBF-1-0076 and detected the encode gene of naphthalene dioxygenase. Based on growth test result, the optimum naphthalene degradationby isolate LBF-1-0076 occured in 75 ppm naphthalene concentration with 15cell concentration. The result of 16S rDNA gene analysis showed that LBF-1-0076 was identified as Shewanella indica strain 0102 with identical value 99%. The result of naphthalene dioxygenase gene detection using Polymerase Chain Reaction (PCR) showed that the isolate contained naphthalene dioxygenase gene with size ±377 bp. Therefore, LBF-1-0076 potential as bioremediation agent to solve crude oil contamination in the sea

Chromatic sensitivity and tinted lenses: A preliminary study with the CAD test

The aim of this study was to assess the extreme effects of tinted lenses on colour vision by examining changes in chromatic sensitivity when viewing visual displays through a slightly tinted (“blue-blocking”) filter and through a heavily tinted, “orange” coloured filter. The CAD test was used to measure both red/green (RG) and yellow/blue (YB) chromatic sensitivity in ten subjects when viewing visual displays through each of the two filters. The measured RG and YB colour thresholds were then compared with similar measurements made without coloured filters in front of the eye. The blue-blocking filter absorbs only a small amount of short-wavelength light, whilst the “orange” filter preferentially attenuates more short-wavelength and some middle-wavelength light. The results show that the blue-blocking filter does not significantly affect either RG or YB colour vision. The orange filter, on the other hand, causes large changes in colour discrimination. The results were statistically analysed by comparing the results obtained with the coloured filters with those measured without any filters in front of the eye. More experimental work is now needed to establish how much short-wavelength light can be removed without significantly affecting the subject’s colour discrimination performance

Generation of the Becker muscular dystrophy patient derived induced pluripotent stem cell line carrying the DMD splicing mutation c.1705-8 T>C

Becker Muscular dystrophy (BMD) is an X-linked syndrome characterized by progressive muscle weakness. BMD is generally less severe than Duchenne Muscular Dystrophy. BMD is caused by mutations in the dystrophin gene that normally give rise to the production of a truncated but partially functional dystrophin protein. We generated an induced pluripotent cell line from dermal fibroblasts of a BMD patient carrying a splice mutation in the dystrophin gene (c.1705-8 T>C). The iPSC cell-line displayed the characteristic pluripotent-like morphology, expressed pluripotency markers, differentiated into cells of the three germ layers and had a normal karyotype

Clinical variability at the mild end of BRAT1-related spectrum: Evidence from two families with genotype–phenotype discordance

Biallelic mutations in the BRAT1 gene, encoding BRCA1-associated ATM activator 1, result in variable phenotypes, from rigidity and multifocal seizure syndrome, lethal neonatal to neurodevelopmental disorder, and cerebellar atrophy with or without seizures, without obvious genotype-phenotype associations. We describe two families at the mildest end of the spectrum, differing in clinical presentation despite a common genotype at the BRAT1 locus. Two siblings displayed nonprogressive congenital ataxia and shrunken cerebellum on magnetic resonance imaging. A third unrelated patient showed normal neurodevelopment, adolescence-onset seizures, and ataxia, shrunken cerebellum, and ultrastructural abnormalities on skin biopsy, representing the mildest form of NEDCAS hitherto described. Exome sequencing identified the c.638dup and the novel c.1395G>A BRAT1 variants, the latter causing exon 10 skippings. The p53-MCL test revealed normal ATM kinase activity. Our findings broaden the allelic and clinical spectrum of BRAT1-related disease, which should be suspected in presence of nonprogressive cerebellar signs, even without a neurodevelopmental disorder

A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum

Tight coordination of gene expression in the developing cerebellum is crucial for establishment of neuronal circuits governing motor and cognitive function. However, transcriptional changes alone do not explain all of the switches underlying neuronal differentiation. Here we unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. The motifs enriched in modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints by directly binding near the 3′ splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, resulting in motor coordination defects, ataxia, and abnormal social behavior. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and establishes motor and cognitive circuitry in the developing cerebellum

Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and removal of intracellular Ca2+. Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD

A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum

Tight coordination of gene expression in the developing cerebellum is crucial for establishment of neuronal circuits governing motor and cognitive function. However, transcriptional changes alone do not explain all of the switches underlying neuronal differentiation. Here we unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. The motifs enriched in modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints by directly binding near the 3′ splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, resulting in motor coordination defects, ataxia, and abnormal social behavior. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and establishes motor and cognitive circuitry in the developing cerebellum

A novel piggybac transposon inducible expression system identifies a role for akt signalling in primordial germ cell migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development