Cloning, expression, and functional characterization of in-house prepared human leukemia inhibitory factor

Leukemia inhibitory factor [LIF] plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF [hLIF] is an essential growth factor for the maintenance of mouse embryonic stem cells [ESCs] and induced pluripotent stem cells [iPSCs] in a pluripotent, undifferentiated state. In this experimental study, we cloned hLIF into the pENTR-D/TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami[TM] 2[DE3] pLacI competent cells to produce the His6-hLIF fusion protein. This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. Our results showed no significant differences in function between laboratory produced and commercialized hLIF

Cloning, expression and functional characterization of in-house prepared human basic fibroblast growth factor

Human basic fibroblast growth factor [bFGF] plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells [hESCs] and human induced pluripotent stem cells [hiPSCs]. In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability. This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo. The results show no significant difference in function between our in-house produced and commercialized bFGF

Evaluation of stability of chitinase gene in transgenic offspring of cotton [Gossypium hirsutum]

Cotton cultivar Coker has been already transformed with recombinant pBI121-chi via Agrobacterium tumefaciens. The T-DNA region of pBI121-chi carries the chitinase [chi] gene from bean and is under the control of the CaMV35S promoter. T1 and T2 progenies of transgenic cotton containing the chi gene were used in this study. Polymerase chain reaction [PCR], Southern and Western blotting data confirmed integration and expression of the chi gene in the T1 and T2 progenies. The growth of Verticillium dahliae was singnificantly inhibited in an in vitro bioassay for which 100 micro g of crude leaf protein extract derived from the T1 plants was used. The 850-bp expected chi fragment was amplified for 77 transgenic plants from 128 T1 and T2 progenies, and 75 transgenic plants showed both chi and nptII bands. T0 conduct bioassay, cotton seedlings were infected with the spore suspension [10[6] spores/ml], in a greenhouse. Fifty-five percent of the transgenic plants were able to restrict V. dahliae growth and symptoms. There were no distinguishable differences in the phenotypic appearance of transgenic plants compared to non-transgenics. These results showed that transgenic cotton expressing a bean chitinase exhibited enhanced resistance against V. dahliae in greenhouse and in-vitro assay as compared to the non-transgenic plants