Genetic reconstitution

Morel L, Croker BP, Blenman KR et al 2000 Genetic reconstitution of systemic lupus erythematosus immunopathology with polycongenic murine strains. Proc Nad Acad Sci USA 97 6670-6675... 

Morel, L., Croker, B.P., Blenman, K.R., Mohan, C., Huang, G., GiUceson, G., and Wakeland, E.K. (2000) Genetic Reconstitution of Systemic Lupus Erythematosus Immunopathol-ogy with Polycongenic Murine Strains, Proc. Natl. Acad. Sci. USA 97, 6670-6675. 

In this experiment, gene expression was assessed by several methods. Liver biopsies found hepatocytes which expressed the LDL receptor. In addition, small changes in cholesterol metabolism were found. However, the clinical effect was negligible and the investigators concluded that the ex vivo method was limited by the low efficiency of genetic reconstitution . 

Numerous factors limited the efficiency of genetic reconstitution. First was the number of cells that were recovered from the liver specimen. A 250-gm liver specimen was resected, and from this only 3 billion hepatocytes were isolated for culture. A typical liver contains approximately 100 million hepatocytes per milliliter and thus the 250-gm specimen could have provided approximately 25 billion hepatocytes. Second, only 2 billion hepatocytes were viable, and only 20% of them expressed the LDL-R after transfection with the retrovirus. Even if these 400 million hepatocytes expressed the normal number of LDL receptors, they would have only the number of receptors typically found in 4 ml of liver. Since the average liver volume is slightly more 1600 ml [24], even if the methods were improved 100-fold, the result would be a LDL receptor levels that would still only be 25% of normal. Cholesterol metabolism is clearly sensitive to the number of LDL receptors since patients with one half the number of LDL receptors (heterozygotes for familial hypercholesterolemia) have markedly elevated levels of LDL cholesterol and develop premature atherosclerosis... 

Meir E, Munro EM, Odell GM, Von Dassow G. Ingeneue a versatile tool for reconstituting genetic networks, with examples from the segment polarity network. J Exp Zool 2002 294 216-51. 

Recently, it has been possible to grow cells of the human immune system in special mice. These mice carry a genetic defect called severe combined immunodeficiency (SCID), which leaves them with crippled immune systems, much like those in AIDS patients. Because SCID mice lack functional cellular immunity, it is possible to implant them with human cells without tissue rejection taking place. Researchers have recently developed techniques to implant human fetal tissues containing stem cells for the blood into SCID mice. It is then possible to reconstitute these mice with functional human immune system cells, including T lymphocytes and B lymphocytes. They have also found that if these SCID mice are infected by HIV, the virus will establish infection in the human tissue and destroy the T helper lymphocytes, just as it does in humans. Thus, it may be possible to study some of the mechanisms by which HIV attacks the immune system in these mice. In addition, they may be very useful for testing potential antiviral drugs. 

There have been two basic approaches. First one involves isolation of the chromatin and nucleosome from the healthy and diseased cell line. The second approach is the reconstitution of the model target such as nucleosome followed by the association with the drug(s). The second approach has been extensively employed to identify the binding site in the protein-nucleic acid complex. A pre-knowledge about the components and their arrangements in the reconstituted system sometime makes it the preferred approach. Different biophysical, biochemical and genetic techniques have been employed to understand the mode of association and the effect of the drugs upon chromatin/nucleosome structure and function. 

Several lines of evidence indicate that CENP-A replaces conventional H3 in the nucleosome. Biochemical studies showed that CENP-A co-sediments with nucleo-some core particles [7] and a genetic analysis indicates an interaction between Cse4p, the CENP-A of Saccharomyces cerevisiae, and H4 [16,17]. A recent study with CENP-A purified from HeLa cells or expressed in bacteria showed that it can substitute for conventional H3 in nucleosome reconstitution [18]. Reconstituted CENP-A-containing nucleosomes appear to contain the other core histones in appropriate stoichiometry. However, they did not strongly protect 146 bp of core DNA from micrococcal nuclease, suggesting that CENP-A may significantly alter some aspects of the core nucleosome structure. 

In vitro/ in vivo Repopulation and differentiation CD34+ cells isolated at day +330 from the BM of an ADA-SCID patient The lymphoid differentia-tion capacity of CD34+ cells was maintained, and genetically corrected HSCs retained their ability to reconstitute lymphopoiesis in a secondary transplant (SCID-hu mice) after infusion. 515130... 

Following Zamecnik s discovery that ribosomes are the complexes responsible for protein synthesis, and following elucidation of the genetic code, the study of ribosomes accelerated. In the late 1960s Masayasu Nomura and colleagues demonstrated that both ribosomal subunits can be broken down into their RNA and protein components, then reconstituted in vitro. Under appropriate experimental conditions, the RNA and protein spontaneously reassemble to form 30S or SOS subunits nearly identical in structure and activity to native subunits. This breakthrough fueled decades of research into... 

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