We have generated transgenic mouse lines expressing exclusively a human INS transgene on an Ins1/Ins2 double knockout (mIKO) background.The transgene expression was driven by either a 4000 bp or a 353 bp promoter.These transgenic lines, designated mIKO:INS 4000 and mIKO:INS 353 , were viable and fertile.Determination of the amounts of insulin transcripts and total pancreatic insulin content revealed relative insulin underproduction in both lines, from birth to adulthood.Total pancreatic insulin stores in mIKO:INS 4000 and mIKO:INS 353 mice represented only about 50% and 27%, respectively, as compared to wild-type mice.Morphometric analysis of pancreas did not show any compensatory betacell hyperplasia.The majority of animals in both lines remained normoglycemic throughout their lives.Nevertheless, glucose tolerance tests revealed glucose intolerance in nearly half of mIKO:INS 4000 male mice, likely due to impaired insulin secretion detected in those animals.In addition, a small fraction (2-4%) of male mice in both lines spontaneously developed diabetes with very distinct pathophysiological features.Diabetes was never seen in female animals.The diabetes developed by mIKO:INS 353 mice was rapidly lethal, accompanied by a dramatic depletion of pancreatic insulin stores whereas the mIKO:INS 4000 diabetic animals could live for several months.This suggests a possible link between the structure of the human INS gene promoter and the type of diabetes developed in these lines.

We report here knock-in of diphteria toxin A chain (dta) gene at the Ins2 locus, using the strategy previously employed to insert lacZ under control of the Ins2 promoter. Mutant Ins2dta/+, Ins2dta/lacZ or Ins2lacZ/+ mouse pups were generated by breeding and analyzed to study the effects of toxigenetic β-cell ablation on islet development and to localize the extrapancreatic Ins2 expression site in the brain. Ins2dta/+ and Ins2dta/lacZ pups developed a severe diabetic ketoacidosis and died rapidly. Histological analysis of their pancreas revealed that β-cells completely disappeared in their islets as evidenced by loss of lacZ activity or insulin immunonostaining. β-cell ablation did not alter the size of other islet cell populations which were normal at birth, although the glucagon-cell population was reduced by 85% at embryonic day E12.5. In the brain, comparative analysis of lacZ expression in Ins2lacZ/+ and Ins2dta/lacZ mice identified the choroid plexus (CP) as a major Ins2 expression site. This finding was confirmed by RT-PCR analysis of insulin transcripts in RNAs prepared from microdissected wild-type CP. Transcripts for other key β-cell markers, with the notable exception of Pdx-1, were also found in CP RNAs. These results must revive interest in studies focused on extrapancreatic insulin gene expression.

The authors have derived a new beta-cell line (betaIns2(-/-lacZ)) from Ins2-/- mice that carry the lacZ reporter gene under control of the Ins2 promoter. betaIns2(-/-lacZ) cells stained positively using anti-insulin antibody, expressed beta-cell-specific genes encoding the transcription factor PDX-1, glucokinase, and Glut-2, retained glucose-responsiveness for insulin secretion, and expressed the lacZ gene. Analysis of Ins1 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Ins1 transcripts were significantly raised to compensate for the lack of Ins2 transcripts in betaIns2(-/-lacZ) cells, as compared to those found in betaTC1 cells expressing both Ins1/Ins2. Thus, transcriptional up-regulation of the remaining functional insulin gene in Ins2-/- mice could potentially contribute to the beta-cell adaptation exhibited by these mutants, in addition to the increase in beta-cell mass that we previously reported. We have also shown that lacZ expression, as analyzed by determining beta-galactosidase activity, was up-regulated by incubating betaIns2(-/-lacZ) cells with GLP-1 and/or IBMX, 2 known stimulators of insulin gene expression. These cells thus represent a new tool for testing of molecules capable of stimulating Ins2 promoter activity.

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic β-cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).

Three transgenic mouse lines, Tg 74, Tg 174, and Tg 171, were obtained by microinjection of an 11-kb human DNA fragment carrying the insulin gene into pronuclei of fertilized mouse eggs. The human insulin gene was expressed in all three transgenic mouse lines as shown by the presence of human C peptide in serum and urine and of human insulin transcripts in RNA prepared from pancreas. Several copies of the human DNA fragment arranged in head-to-tail arrays were present in each line. The human DNA insert was transmitted to the progeny as a single genetic locus. The chromosomal integration of the human insulin transgene was directly demonstrated by in situ hybridization to metaphase chromosomes of mitotic cells prepared from spleen and bone marrow. The insert appeared unique and located on a different chromosome in each line, namely 7 for Tg 74, 13 for Tg 174, and 18 for TG 171. Separation of DNA fragments larger than 20 kb by pulse-field electrophoresis showed that several insertion sites were present in each chromosome locus. This is the first direct evidence in transgenic mice that a gene located at various chromosome loci can be correctly expressed.

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2 microglobulin (β2m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse β2-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human β2m restores these reactivities. Since most of the amino acid differences between mouse and human β2m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.

The state of intracellular viral DNA in cells infected with channel catfish virus has been studied by the Hirt selective extraction procedure and by restriction endonuclease digestion. The sedimentation properties and restriction patterns of viral DNA in the Hirt supernatant fraction indicate that the majority, if not all, of the DNA is in the form of linear unit-length (Mr approximately equal to 85 x 10(6)) molecules. However, restriction digests of viral DNA in the pellet fraction lacked two fragments corresponding to the molecular ends of unit-length DNA. In addition, there appeared in HpaI digests of pellet DNA a new restriction fragment interpretable as the product of fusion between the ends of unit-length molecules. The size of the new fragment requires that fusion occur in such a way that one copy of the terminally repeated sequences (Mr approximately equal to 12.3 x 10(6)) of the unit-length DNA is lost in the process. In pulse-chase experiments, radioactivity flowed from the pellet fraction to the supernatant fraction, suggesting a precursor-product relationship for these DNA species. The results are easily understood if unit-length virion DNA is generated by excision from concatemeric structures.

The drug 2,3-bis(acetyl mercaptomethyl)-quinoxalin (BAMMQ) was 99.9% inhibitory for herpes simplex virus (HSV) multiplication in cell cultures at concentrations of 1.6 X 10(-5) M or less. The drug was not inactivating for the virus, did not interfere with adsorption and penetration of the virus, and was still active when added as late as 12 h after infection with HSV. BAMMQ did not prevent HSV DNA replication, but interfered with a late stage of virus assembly and/or maturation.

A variety of derivatives from quinoxaline were tested for their antiviral properties. One of them, 2,3-bis-(acetylmercaptomethyl)-quinoxaline (BAMMQ), inhibited poliovirus growth by 99.8% or more at concentrations as low as 10∼5 M. Poliovirus RNA replication was arrested within 30–60 min after addition of the drug. Poliovirus protein synthesis was not directly affected. The drug was also found to be inhibitory for the in vitro synthesis of poliovirus RNA.

SUMMARY CV1, BSC1, Vero, and primary monkey kidney cells were seeded either at low cell density and infected with poliovirus while growing, or at high cell density and infected under conditions of contact inhibition. Virus RNA synthesis and virus production were delayed in the cells seeded at high cell density, although neither adsorption nor uncoating of the parental virus particles were altered. In spite of the delay, however, virus RNA synthesis, once initiated, proceeded in the same fashion and at the same rate in both growing and resting cells. This implies that one of the first events following uncoating of the infecting virus was temporarily retarded in resting cells. There are indications that this delay might be due to a lag in the formation of parental virus polyribosomes.

The thermosensitive defect of the Sabin LSc2ab strain of poliovirus type I was studied. Transfer of infected KB cells from 36 to 38.5 C resulted in 30% inhibition of viral RNA replication but in 90% inhibition of formation of virions. Neither 74S procapsids nor 14S particles were detected in the cells transferred to the non-permissive temperature. However, procapsids, once accumulated at 36 C, were normally stable at 38.5 C and could transform into virions at that temperature. Viral proteins synthesized at the nonpermissive temperature were not different from those synthesized at permissive temperature, as judged from their pattern in polyacrylamide gel electrophoresis and from the fact that they normally matured into virions when the infected cells were brought back to permissive temperature, even under conditions of inhibition of protein synthesis. This leads to the conclusion that the defect in the Sabin strain studied lies in the assembly of its viral capsid proteins into capsomeres.

Sodium dodecyl sulfate was found to aggregate virions from the Sabin Lsc2ab strain of poliovirus type 1. Aggregation was prevented by high ionic strength buffers. A procedure is described for the rapid purification of the virus through the use of sarkozyl.

Summary In HeLa cells infected with poliovirus, shifts from optimal to supraoptimal temperatures in the middle of the infectious cycle resulted in extensive degradation of newly made virus RNA. This temperature-induced degradation was not observed until about 3 hr after infection. It did not require simultaneous synthesis of proteins. During the first 3 hr of infection at 39·5°, RNA of temperature-resistant poliovirus strains replicated much faster than at 36°. Subsequently, degradation became predominant. Thus, temperature-resistant strains partially escaped the effects of degradation by faster replication. During the first 150 min. approximately, the RNA of temperature-sensitive strains replicated as well at supraoptimal as at optimal temperatures. Subsequently, newly made virus RNA was degraded. The inhibitory effect of supraoptimal temperatures may be due to release, or activation, of a ribonuclease.