Kevin L. Bentley

ProxTom Lymphatic Vessel Reporter Mice Reveal Prox1 Expression in the Adrenal Medulla, Megakaryocytes, and Platelets

Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

RECOMBINOGENIC TARGETING : A NEW APPROACH TO GENOMIC ANALYSIS : A REVIEW

Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of large-insert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems.

Transgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro. The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st (Chst4) gene with flanking sequences. Transgenic mice were generated with the β-galactosidase (LacZ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LTβR-Ig (lymphotoxin-β receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity.

Lymphatic Vessel Function in Head and Neck Inflammation

BACKGROUND Serious infections of the head and neck cause lymphedema that can lead to airway compromise and oropharyngeal obstruction. Lymphangiogenesis occurs in the head and neck during infection and after immunization. The goal of this project was to develop tools to image lymphatic vessels in living animals and to be able to isolate individual lymphatic endothelial cells in order to quantify changes in single cells caused by inflammation. METHODS The ProxTom transgenic red-fluorescent reporter mouse was developed specifically for the purpose of imaging lymphatic vessels in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in development and for the maintenance of lymphatics in adulthood. Mice were immunized and their lymphatic vessels in lymph nodes were imaged in vivo. Individual lymphatic endothelial cells were isolated by means of their fluorescence. RESULTS The ProxTom transgene has the red-fluorescent reporter td-Tomato under the control of Prox1 regulatory elements. tdTomato was faithfully expressed in lymphatic vessels coincident with endogenous Prox1 expression. We show lymphangiogenesis in vivo after immunization and demonstrate a method for the isolation of lymphatic endothelial cells by their tdTomato red-fluorescence. CONCLUSIONS The faithful expression of the red-fluorescent reporter in the lymphatic vessels of ProxTom means that these mice have proven utility for in vivo study of lymphatic vessels in the immune response. ProxTom has been made available for distribution from the Jackson Laboratory: http://jaxmice.jax.org/strain/018128.html .

A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes

Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large‐insert DNAs representing genes of interest. We have developed a methodology using a yeast‐bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)‐sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette‐mediated capture of large‐insert genomic fragments from BACs‐human polycystic kidney disease‐1 (PKD1); modification of pClasperA clones by the RareGap method‐PKD1 mutations; Claspette‐mediated modification of pClasper clones—mouse albumin‐1 gene; and, of most relevance to our interest in lymph node vasculature—Claspimer‐mediated modification of pClasper clones‐high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.

High Endothelial Venule Reporter Mice to Probe Regulation of Lymph Node Vasculature

Lymphotoxin (LT) is crucial for the regulation of HEV adhesion molecules MAdCAM-1 and PNAd and a sulfotransferase, GlcNAc6ST-2 (gene symbol Chst4); here called HEC-6ST. Following immunization, some HEVs express markers of both HEVs (PNAd) and LVs (LYVE-1). In order to evaluate this process in real time, we have developed mice transgenic for a construct that consists of an HEV specific gene driving a green fluorescent reporter gene (eGFP). These mice express the reporter gene in HEVs in concurrence with the endogenous gene and PNAd. Additional mice transgenic for lymphatic vessel reporter constructs are in development. These will provide material for in vivo imaging and allow us to evaluate the regulation and interaction of HEVs and LVs.

A Transgenic Mouse with Red-Fluorescent Lymphatic Vessels:

ture) began to enlarge. Under general anesthesia, the right side of the neck was opened through the previous laryngectomy incision. A plane of dissection was established between the trachea and the esophagus and they were separated. The tracheal and esophageal components of the fistula were each closed primarily with Vicryl sutures. An inferiorly-based right sternocleidomastoid muscle flap was interposed between the trachea and esophagus. After 6 weeks, a secondary tracheoesophageal puncture was performed and an 8mm Provox 2 prosthesis inserted with excellent fit and immediate restoration of the voice. RESULTS: No further leakage has occurred to date. CONCLUSION: An effective technique is described for dealing with the uncommon but difficult problem of peri-prosthetic leakage after surgical voice restoration.