An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G 2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.
Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O 1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre-formed sulfate-binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfatebinding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.
The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.
Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin ␣v3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of ␣v3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.
Foot-and-mouth disease virus (FMDV) capsids are inherently labile under mildly acidic conditions, dissociating to pentamers at pH values in the region of 6n5, with the release of protein 1A and the viral RNA. This acid-induced disassembly is thought to be required for the entry of the virus genome into the host cell. Previous work has highlighted a histidine-α-helix charge-dipole interaction at the twofold axes of symmetry between pentamers and has suggested that this interaction plays a role in acid-induced disassembly. The validity of this theory has now been tested by converting the implicated residue, His-142 of protein 1C, to Arg, Phe and Asp. The effects of such changes were studied by using a previously described vaccinia virus expression system, in which synthesis and processing of FMDV capsid proteins results in the self-assembly of capsids. In agreement with the histidine-α-helix charge-dipole theory, assembly in the arginine mutant was found to be greatly reduced, while capsids of the aspartic acid mutant were considerably more stable under acidic conditions than the wild-type. Aberrant but acid-stable complexes were obtained in the phenylalanine mutant.
Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.
The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular alpha(v)beta3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of alpha(v)beta3 was exquisitely sensitive to competition by short RGD-containing peptides (50% inhibition at < 10(-8) M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10(4) fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to alpha(v)beta3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for alpha(v)beta3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.
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