Abstract
The creation of knockout animals is frequently complicated by embryonic lethality or infertility. Moreover, since their creation relies on the use of embryonic stem cells from strain 129 mice, all knockout mice are originally on a homozygous or heterozygous 129 background. Investigators in Taiwan have developed a novel system for the creation of viable transgenic mice that can be induced to silence genes of interest in specific target organs at will. The approach combines interference RNA (siRNA) for specific gene knockdown, CreloxP for tissue-specific expression, and tetracycline-on for inducible expression. One vector contains the sequence encoding an siRNA that is expressed only after Cre-mediated recombination. The other vector expresses the Cre recombinase under the control of the tissue-specific promoter and doxycycline. Each vector is used to create a stably transgenic line of animals and the two lines are mated to obtain doubly transgenic animals. Administration of doxycycline induces tissue-specific Cre expression. This, in turn, drives rearrangement and activation of the siRNA vector in the same cells.
Chang HS, Lin CH, Chen YC, Yu WC: Using siRNA technique to generate transgenic animals with spatiotemporal and conditional gene knockdown. Am J Pathol 165:1535–1541, 2004
Successful long-term administration of exogenous therapeutic proteins such as erythropoietin can be both costly and challenging. A new gene therapy approach to this problem was recently described by a group of British researchers. These investigators created mice transgenic for the human erythropoietin under the control of an immunoglobulin promoter, then immunized the mice with phycoerythrin. Phycoerythrin-specific B cells were isolated and adoptively transferred to syngeneic animals. When stimulated with phycoerythrin, the transferred lymphocytes proliferated, produced human erythropoietin, and corrected anemia in the recipient mice. This demonstrates that memory lymphocytes, which are very long-lived and undergo clonal expansion when exposed to specific antigen, can secrete cloned proteins with biological activity. This may offer a new approach for therapeutic protein delivery.
Takacs K, Du Roure C, Nabarro S, Dillon N, McVey JH, Webster Z, Macneil A, Bartok I, Higgins C, Gray D, Merkenschlager M, Fisher AG: The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes. Proc Natl Acad Sci U S A 101:16298–16303, 2004
A consortium of over 100 international scientists recently completed the draft genome sequence of the red jungle fowl, Gallus gallus, the ancestor of the domestic chicken. The chicken genome is one-third the size of most mammalian genomes. The small size of the genome appears to be due to the paucity of retroposed pseudogenes, the absence of short interspersed nucleotide elements, and reduced large segmental duplications. The chicken has one pair of sex chromosomes and 38 autosomes. Autosomes include large macrochromosomes that resemble mammalian chromosomes and tiny microchromosomes not commonly found in mammals. Microchromosomes are GC-rich and contain few repetitive sequences. Comparison of the chicken and human genomes indicates that at least 70 megabases of shared sequence are likely to be functional in both species. These findings will provide information valuable for the fields of agriculture and comparative genomics.
International Chicken Genome Sequencing Consortium: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432:695–716, 2004
Epithelial tumors commonly metastasize first to regional lymph nodes, suggesting that lymph nodes pose mechanical or biological barriers to tumor spread. To test this hypothesis, Japanese scientists injected fluorescently labeled syngeneic cancer cells into rat cecum submucosa. They monitored the appearance of these cells in the regional mesenteric lymph node and the response of the lymph node to the cells. Cancer cells were arrested in the marginal sinus of the node. Hyperplasia of lymphoid follicles and medullary lymphoid cells developed very soon after tumor cell arrival, and macrophages migrated into the marginal sinus, where they apparently engulfed apoptotic tumor cells. Depleting lymph nodes of macrophages significantly enhanced the growth of metastatic tumor cells. The cytokines tumor necrosis factor-α, interleukin 1-β, and interleukin 2 also increased in the lymph node after the tumor cells entered the lymph node, while interferon-γ, interleukin-4, and interleukin-12 decreased. These studies demonstrate that lymph nodes do serve, at least transiently, to filter metastatic cancer cells from the circulation.
Nagata H, Arai T, Soejima Y, Suzuki H, Ishii H, Hibi T: Limited capability of regional lymph nodes to eradicate metastatic cancer cells. Cancer Res 64:8239–8248, 2004
Lambs derived by nuclear cloning often develop renal defects, including hydronephros consistent with urinary out-flow obstruction. Nephrogenesis occurs by the reciprocal induction of epithelial and mesenchymal elements. Many of the epithelial elements in the kidneys arise by the process of mesenchymal to epithelial transition. British investigators have now shown that this process is faulty in cloned lambs. By immunohistochemistry, they showed that, compared to normally developing kidneys, cytokeratin expression is increased in damaged tubules; desmin, vimentin, and smooth muscle actin are expressed in undifferentiated mesenchyme; and vimentin and desmin are re-expressed on proximal tubular epithelial cells in the kidneys of cloned lambs. The etiology of this dysregulated developmental process is not known.
Dawson AJ, King TJ, Wilmut I, Harkness LM, Kelly BG, Rhind SM: Immunohistochemical characterization of cloned lamb nephropathy. J Histochem Cytochem 52:1657–1664, 2004