Retinoblastoma protein purification and transduction of retina and retinoblastoma cells using improved alphavirus vectors.

PURPOSE: To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells. METHODS: Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells. RESULTS: The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines. CONCLUSIONS: This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions.

Thermal assessment of 40-MHz ultrasound at soft tissue-bone interfaces.

Tissue exposure to diagnostic ultrasound (US) can cause significant temperature rises. However, little has been reported on thermal effects of high-frequency US, and guidelines for the use of US do not necessarily apply to higher frequencies. Temperature rise induced by US biomicroscopy (UBM) was measured in phantoms containing mouse skulls and in anesthetized mice and mice post mortem, with a 50-microm K-type thermocouple. The operating frequency was 40 MHz with a free field I(SPTA) of 2.6 mW/cm(2) (B-mode) and 11.9 W/cm(2) (Doppler). Peak negative pressures were 5.22 MPa (B mode) and 7.32 MPa (Doppler), resulting in a mechanical index (MI) of 0.83 (B-mode) and 1.05 (Doppler mode). In Doppler mode, mean temperature rises of 1.80 degrees C and 1.73 degrees C were measured for proximal and distal skull phantom surfaces after a 3-min insonation. In vivo, the proximal mouse skull surface showed a mean temperature rise of 2.1 degrees C, with no statistically significant differences post mortem. Our results indicate temperature rise from insonation of bone interfaces using similar exposure parameters should not cause adverse bioeffects.

In vivo imaging of embryonic development in the mouse eye by ultrasound biomicroscopy.

PURPOSE: New imaging tools now provide an unprecedented opportunity to visualize anatomic and functional development of the mouse eye. In this study, normal embryonic development of the mouse eye was studied by ultrasound biomicroscopy (UBM), with a focus on the formation of the retina, lens, and cornea. METHODS: The growth of 65 embryonic eyes from timed-pregnant CD-1 mice was examined at various stages of development between embryonic day (E)11.5 and E18.5, using 40-MHz UBM. RESULTS: The morphogenesis of ocular tissues including the lens, retina, and orbit were revealed from the earliest stages of development. The major axis of the CD-1 lens grows at a rate of 68 micro m/d, whereas that of the globe grows at a rate of 122 microm/d, with a concomitant exponential increase in volume. CONCLUSIONS: UBM allows noninvasive assessment of ocular morphogenesis in vivo and can be used to calculate relative growth rates of ocular structures.

Genomic amplification in retinoblastoma narrowed to 0.6 megabase on chromosome 6p containing a kinesin-like gene, RBKIN.

All retinoblastomas (RBs) show genomic changes in addition to loss of both RB1 alleles. Quantitative-multiplex PCR analysis identified in 41 of 70 (59%) RBs a 0.6-Mb minimal region of chromosome 6p22 gain from which we cloned the human kinesin gene, RBKIN/KIF13A, by rapid amplification of retinal cDNA. RBKIN is expressed ubiquitously in adult tissues, at low levels in fetal tissues, and at high levels in RB. Antisense RBKIN oligonucleotide reduced the growth rate of RB cell lines. RBKIN and/or another closely linked gene are candidate oncogenes contributing to malignant progression of RB.