Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.     

Highly sensitive and rapid gene mapping using miniaturized blot hybridization: application to prenatal diagnosis

We have developed a protocol for the preparation and analysis of amniocyte DNA which permits more sensitive and more rapid antenatal detection of sickle-cell anemia (SCA) than previously has been possible. After rapid extraction of DNA from amniotic cells, only 50 ng of MstII-digested DNA need be analyzed by mini-gel electrophoresis and hybridization detection to determine reliably the fetal genotype. Under these conditions, the entire gene-mapping procedure can be performed within 5 days. When larger amounts of DNA ($\text{> 500}\text{ng}$) are analyzed, the minimal diagnosis time is reduced to 2 days. The resolution of restriction fragments on mini-gels is comparable to that obtained with larger gels. The 1.15-kb β^A and 1.35-kb β^SMstII fragments are well separated. The technique is useful whenever rapid and sensitive analysis of genomic DNA is desired.