We have identified a short segment of the mouse Pax-6 gene 5' flanking region that is necessary and sufficient for reporter construct expression in components of the eye derived from non-neural ectoderm. This transcriptional control element has a highly conserved nucleotide sequence over 341 bp and is located approximately 3.5 kb upstream of the start-point for transcription from the most proximal promoter (PO) of the Pax-6 gene. The level of identity between the human and mouse Pax-6 genes in this region is 93%. When combined either with its natural promoter or a heterologous minimal promoter, this element directs reporter construct expression to a region of surface ectoderm overlying the optic cup beginning at E8.5-9.0 (12-14 somites). Subsequently, expression is restricted to the lens (primarily the lens epithelium) and the corneal epithelium. This element will provide an important tool in future transgenic analyses of lens formation and will allow identification of transcription factors with a central function in lens development.

Electrotransformation also known as electroporation is the most reliable and efficient tool for plasmid DNA uptake. Electrotransformation efficiency is function of many factors which include (1) number of cell washes prior to electroporation, (2) electroporation cell number, (3) electroporation DNA amount, and (4) cell growth phase. Those factors have limitedly been concomitantly investigated in E. coli DH10B strain. This study is aimed to explore above key factors to define the optimal conditions for high electrotransformation efficiency. The results showed that electrotransformation efficiency of E. coli DH10B was enhanced to 1.5 x 10 9 cfu/µg by washing cells three times with 15 ml of 10% glycerol. This washed off extra salts from cell suspension and enhanced electrotransformation by preventing arcing and enhancing cell resistance while ensuring minimal level of conductivity. Early exponential phase at 0.15 OD600 was the best growth phase for enhancing electrotransformation of E. coli DH10B. The results also showed that higher electrotransformation efficiency was similarly achieved when 0.5 x 10 10 and 0.6 x 10 10 cell numbers were electroporated with DNA amount ranging from 10 to 40 pg. This study confirmed the optimal conditions for electro competent cell preparation and plasmid DNA electrotransformation, which can result highest transformation efficiency.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic * Corresponding Author: smjay@umd.edu. ORCID: Steven M. Jay: 0000-0002-3827-5988The authors declare the following competing financial interest(s): S.M.J. holds a patent related to multivalent ligand technology (US 9,029,328 B2). Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharma-ceut. 6b00919. DNA and protein sequences for affibody constructs, purified affibody mass spectra, surface plasmon resonance data, and immunoblot quantification (PDF) Graphical Abstract HHS Public Access

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted 7 To whom correspondence should be addresseds.