Cinquin Lab

Albazerchi A., Cinquin O., Stern C.D. BMC Dev. Biol. 7:25 (2007)

Abstract

BACKGROUND: The mouse anterior visceral endoderm (AVE) and the chick hypoblast are thought to have homologous roles in the early stages of neural induction and primitive streak formation. In mouse, many regulatory elements directing gene expression to the AVE have been identified. However, there is no technique to introduce DNA into the chick hypoblast that would enable a comparison of their activity and this has hampered a direct comparison of the regulation of gene expression in the mouse and chick extraembryonic endoderm. RESULTS: Here we describe a new method to introduce DNA into the chick hypoblast, using lipofectamine-mediated transfection. We show that the hypoblast can be easily transfected and that it starts to express a luciferase reporter within 2 hours of transfection. The validity of technique is tested by following the movement and fate of hypoblast cells, which reveals their translocation to the anterior germinal crescent. We then introduce a vector containing GFP driven by the mouse VEcis-Otx2 enhancer (which directs gene expression to the mouse AVE) and we detect activity in the hypoblast. CONCLUSION: The new technique for delivering expression constructs to the chick hypoblast will enable studies on gene activity and regulation to be performed in this tissue, which has proved difficult to transfect by electroporation. Our findings also reveal that regulatory elements that direct gene expression to the mouse AVE are active in chick hypoblast, supporting the idea that these two tissues have homologous functions.

Cinquin O., Christopherson R.I., Menz R.I., Mol. Biochem. Parasitol. 117(2), pp245-247 (2001)

Abstract

The RIG plasmid [Baca and Hol, 2000, Int. J. Parasitol. 30, 113-118] encodes tRNA genes rare in Escherichia coli, enabling codon-biased Plasmodium genes to be over-expressed in E. coli. Expression of orotidine-5′-monophosphate decarboxylase (EC 4.1.1.23) from Plasmodium falciparum was found to be toxic to E. coli BL21(DE3) cells carrying the RIG plasmid. Unfortunately, the RIG plasmid is incompatible with the pLysS plasmid commonly used with pET expression vectors to allow over-expression of recombinant proteins toxic to E. coli by repressing their basal level expression. We describe here a new hybrid plasmid carrying the gene for T7 lysozyme and those for the rare E. coli codons argU, ileX and glyT, which facilitates over-expression of orotidine-5′-monophosphate decarboxylase (EC 4.1.1.23) from P. falciparum.