We thank L. Dabo for assistance with mouse genotyping and A. McArthur for editing the manuscript.

No abstract

Background: The RB1 gene was the first tumor suppressor gene cloned from humans by studying genetic lesions in families with retinoblastoma. Children who inherit one defective copy of the RB1 gene have an increased susceptibility to retinoblastoma. Several years after the identification of the human RB1 gene, a targeted deletion of Rb was generated in mice. Mice with one defective copy of the Rb gene do not develop retinoblastoma. In this manuscript, we explore the different roles of the Rb family in human and mouse retinal development in order to better understand the speciesspecific difference in retinoblastoma susceptibility.

Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate-specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID™-Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID™-Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross-react with other human or nonhuman tissues tested. RSID™-Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA-based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID™-Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.

Current methods for forensic identification of saliva generally assay for the enzymatic activity of alpha-amylase, an enzyme long associated with human saliva. Here, we describe the Rapid Stain IDentification (RSID-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID-Saliva detects less than 1 microL of saliva. The sensitivity of RSID-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.

Rb1 is essential for normal embryonic development, as null mice die in midgestation with widespread unscheduled cell proliferation. Rb1 protein (pRb) mediates cell cycle control by binding E2F transcription factors and repressing expression from E2F-dependent promoters. An increasing amount of evidence suggests that pRb loss also compromises cellular differentiation. Since differentiation is often dependent on cell cycle exit, it is currently unclear whether the effects of pRb on differentiation are an indirect consequence of pRb/E2F-mediated cell cycle control or whether they reflect direct cell-type-specific pRb functions. We have mutated Rb1 in the mouse to express a protein (R654W) specifically deficient in binding E2F1, E2F2, and E2F3. R654W mutant embryos exhibit cell cycle defects the same as those of Rb1 null embryos, reinforcing the importance of the interactions of pRb with E2F1, E2F2, and E2F3 for cell cycle control. However, R654W embryos survive at least 2 days longer than Rb1 null embryos, and increased life span is associated with improved erythrocyte and fetal liver macrophage differentiation. In contrast, R654W pRb does not rescue differentiation defects associated with pRb-deficient retinae. These data indicate that Rb1 makes important cell-type-specific contributions to cellular differentiation that are genetically separable from its general ability to stably bind E2F1, E2F2, and E2F3 and regulate the cell cycle.

With sexual assault evidence, the visualization of spermatozoa confirms that ejaculation has occurred. However, microscopic examination of spermatozoa is a laborious process and can sometimes result in sperm cells being overlooked. Here, we present the developmental validation of the SPERM HY-LITER™ kit, which contains a human sperm-specific mouse monoclonal antibody coupled to a fluorescent Alexa 488 dye. The kit was tested using samples of human semen, saliva, blood, and urine, various animal semen extracts, sexual lubricants, and a commercially available spermicidal film. Postcoital vaginal swabs, degraded semen samples, and samples prepared with sample fixation techniques that deviated from the kit-provided protocol were also tested. In each case, the SPERM HY-LITER™ kit was demonstrated to bind only to human sperm cell heads. Limitations to this fluorescent staining procedure include nonspecific staining and increased background fluorescence with extreme heat fixation in some samples.

In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression.