Ann Boyd

CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures

Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

Activation of mouse retrovirus by herpes simplex virus type 1 cloned DNA fragments

Abstract Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the ∼3.4-kilobase pair (kbp) Bam HI fragment which contains the virus thymidine kinase (TK) gene, and the ∼5.3-kbp Eco RI L fragment. Activation by the TK-containing Bam HI fragment was abrogated by digestion with Eco RI. Activation within the S region of HSV-1 DNA was observed with the ∼15.2-kbp Eco RI H ragment and the ∼8.4-kbp Eco RI/ Hin dIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.

Functional mapping of the Epstein-Barr virus genome: Identification of sites coding for the restricted early antigen, the diffuse early antigen, and the nuclear antigen

Attempts were made to functionally map antigenic expression of the Epstein-Barr virus (EBV) to specific regions on the EBV genome, using the B95-8 strain. Experiments were performed to map the expression of early antigen (EA), both restricted and diffuse (R and D, respectively), and the EBV nuclear antigen (EBNA), using intact B95-8 DNA, cloned BamHI fragments or Charon 4A fragments. DNA preparations were microinjected into two EBV genome-negative epithelial tumor cell lines. Expression of EBV antigens was monitored using precharacterized human sera, as well as monoclonal antibodies to EA-R and EA-D. The data suggest that EA-R maps to the BamHI H fragment, and EA-D maps to the Charon 4A fragment 7. A previous report that BamHI K is associated with the expression of a nuclear neoantigen tentatively identified as EBNA (W.P. Summers, E.A. Grogan, D. Shedd, M. Robert, C.R. Liu, and G. Miller, Proc. Nat. Acad. Sci. USA 79, 5688-5692, 1982) was also confirmed.

Cooperativity of SV40 T antigen and ras in progressive stages of transformation of human fibroblasts

Human diploid fibroblasts immortalized by SV40 T antigen provide an experimental system for studying the progression and synergism in transformation by secondary oncogenes. We have utilized the human fibroblast line HAL, which was immortalized with an orgin-defective SV40 genome encoding a temperature-sensitive T antigen, to study the cooperativity between SV40 T antigen and the ras oncogene in the progression of transformation. This study demonstrates that HAL cells possess characteristic growth patterns, requiring 10% serum, are anchorage dependent, and express a temperature-sensitive T antigen. HAL cells rely on the normal functioning of T antigen for continual growth and therefore do not proliferate at 39 degrees C. Three new derivatives of the HAL cell line were generated by microinjection of the ras oncogene. The cell line v-ras-HAL was derived by microinjection of HAL cells with v-Ha-ras DNA. The cell lines c-rasSVneo-HAL and c-rasLTRhygro-HAL were established by microinjection of HAL cells with the plasmids pSV2neoT24 or fpHVT24, respectively, wherein the ras gene is transcriptionally regulated by the cellular promoter and driven by either the SV40 enhancer or an upstream LTR enhancer. The three ras containing cell lines grow in reduced serum concentrations (0 to 5%), are anchorage independent, and express both T antigen and ras p21. The v-ras-HAL and c-rasSVneo-HAL cell lines are still dependent upon the normal functioning of T antigen for continual growth at 39 degrees C, however the c-rasLTRhygro-HAL cell line does proliferate at 39 degrees C in 10% serum-containing medium. Therefore, we propose that neither v-Ha-ras nor c-ras can replace T antigen at 39 degrees C; rather T antigen and ras cooperate in progressive stages of transformation of human fibroblasts.

Expression of cloned genes microinjected into cultured mouse and human cells

Abstract The direct approach of capillary microinjection was used to study exogenous gene expression in mouse and human cells. The efficiencies of biochemical and morphological transformation in mouse cells and the differential ras gene expression in mouse and human cells were determined. Biochemical transformation efficiency of mouse cells after microinjection was ≈2.5% with the thymidine-kinase plasmid and ≈30% with the neomycin-resistant gene, pSV2 neo, which contains the SV-40 enhancer sequence. Mouse NIH3T3 cells microinjected with genes of the ras family—including viral isolates, the human bladder oncogene EJ, and N- ras —produced morphologically transformed foci with an efficiency of 10–20%, whereas the normal human cellular homologue of the ras gene in Harvey sarcoma virus showed an efficiency of 0.5–1%. Expression of v- ras genes was compared in mouse and human cells by both immunofluorescence detection of the ras protein, p21, 24 hr after microinjection and by the focus assay. The data suggest that microinjection is a useful approach for the investigation of early exogenous gene expression and control in eukaryotic cells and has the potential to allow insight into oncogenic events.

Expression of type C virus p30 in mouse cells infected with herpes simplex virus.

Abstract Expression of type C virus p30 in BALB/c and NIH Swiss mouse cells infected with live (productive infection) or uv-irradiated herpes simplex virus (uv-HSV) types 1 and 2 was studied by use of either immunofluorescent (FA) or radioimmunoassay (RIA) procedures. No evidence for enhanced expression of p30 was obtained with either of these procedures although activation of type C virus in uv-HSV-infected BALB/c cells was readily demonstrated at low frequencies (∼10 −4 ) by infectious center assay. While FA staining with rabbit anti-murine leukemia virus (MuLV) p30 serum was observed in mouse cells productively infected with HSV or infected with uv-HSV, this was shown to be nonspecific. It was concluded that activation of type C virus in uv-HSV-infected BALB/c cells does not occur in significantly more cells than detected by infectious center assay.

Exogenous DNA expression in eukaryotic cells following microinjection

Microinjection of nucleic acids, DNA, RNA, proteins, and any soluble material into living eukaryotic cells makes it possible to design experiments focused on single cells. In contrast facilitated transfer protocols requires hundreds of thousands of cells from which the expressed gene or intracellular effect must be detected within the culture. In addition to the immediate observable nature of the expressed product and intracellular reaction, microinjection bypasses the uptake toxicity associated with facilitated transfer of foreign material into cultured cells. The direct injection of material into the nucleus or cytoplasm allows the number of treated cells to be monitored and expression efficiencies to be observed directly. Microinjection of a hundred cells grown on small glass coverslips and subsequently counted for expression of the foreign material determines expression efficiency as a percentage of cells injected. The efficiency is based on detection of the foreign inserted gene product and does not control for relative promoter efficiency between constructs. The purpose is not to compare two constructs to each other but to monitor dual expression. The creation of marker fluorescent proteins, such as the green fluorescent protein (GFP) in the same expression plasmid with a test gene allows the immediate observation of the GFP injected cells and within the same cells the positive or negative expression of the test gene. Expression of a foreign gene, such as SV40 T antigen cloned into an expression vector can be detected four hours after microinjection of the DNA. Fusing GFP into the same expression region of the T coding sequence labels T-GFP as a fusion protein with characteristic T immunological staining nuclear patterns but allows the cells to be studied without fixation through sequential periods of observation. The direct nature of microinjection allows comparison of gene expression in a variety of cells and the determination of the number of cells expressing the exogenous material in relationship to the number of cells injected.

Identification of EBV-Specific Antigens Following Microinjection of Subgenomic DNA Fragments

The regions of the Epstein-Barr Virus (EBV) genome which code for proteins within the early antigen (EA) and viral capsid antigen (VCA) complex were identified by indirect immunofluorescence (IF) 2–4 days after microinjection of subgenomic cloned fragments of EBV DNA. Two new regions have been identified as part of the early antigen (EA) complex, namely, the Charon 4A cloned fragments which cover map units 38–47 and 83–93 respectively. One DNA fragment from map units 45–54, produces a protein in human cells after microinjection which reacts with EA-VCA+ human sera. Attempts to transform human B-lymphocytes from cord blood with a variety of EBV DNA fragments is described.

The New Program of the World Health Organization in Medical Virology

The World Health Organization (WHO) convened a Scientific Group to adapt its program in virus diseases to recent progress in virology. The program consists of (a) general activities, such as reference services and the supplying of reagents by the WHO Collaborating Centres and (b) specific activities to solve problems-including the promotion of necessary research-caused by certain diseases of public health importance. The Group reviewed problems caused by influenza and other respiratory viruses, enteroviruses, gastroenteritis viruses (for which types A and B have been proposed as a convenient nomenclature), viral hepatitis, viruses in water and sewage, arboviruses, arenaviruses and Marburg virus, measles and rubella vaccination, smallpox, rabies, chronic infections, herpesviruses, oncogenic viruses, congenital infections, nosocomial infections, chlamydial and rickettsial infections, and mycoplasma infections.

Characterization of Single-Cell Clonal Lines Derived from HSV-2-Transformed Mouse Cells

A BALB/c continuous cell line, 238, was transformed following infection with UV-inactivated herpes simplex virus type 2 (HSV-2) was designated H238. Several cell lines were derived from single cells of H238 by cloning in soft agar. The clonal lines express cytoplasmic antigens by immunofluorescence when reacted with rabbit HSV-2 antiserum and mouse tumor-bearer serum. The cloned cell lines induce tumors in immunocompetent mice by the subcutaneous route with a 100% incidence at an inoculum of 10(6) cells. These properties are consistent with those of the H238 parent cell line. In contrast to the parent line, however, the cloned cell lines do not elicit HSV neutralizing antibody in the tumor-bearing host.