Pre-B-cell development in the absence of λ5 in transgenic mice expressing a heavy-chain disease protein

AbstractBackground: Heavy-chain diseases (HCDs) are human lymphoproliferative neoplasias that are characterized by the secretion of truncated immunoglobulin heavy chains devoid of light chains. We have previously proposed — by analogy to the process by which mutated growth factor receptors can be oncogenic — that because the genetic defects in HCDs result in the production of abnormal membrane-associated heavy chains lacking an antigen-binding domain, these abnormal B-cell antigen receptors might engage in ligand-independent signalling. Normal pre-B-cell development requires the presence of the pre-B-cell receptor, formed by the association of μ heavy chains with two polypeptides — so-called surrogate light chains, Vpre-B and λ5 — that are homologous to the variable and constant portions of immunoglobulin light chains, respectively. To assess whether amino-terminal truncation of membrane-associated heavy chains results in their constitutive activation, we have examined the ability of a HCD-associated μ protein to promote pre-B-cell development in transgenic mice.Results When the μ HCD transgene is introduced into SCID mice, CD43− pre-B cells develop normally. To determine whether this pre-B-cell development requires surrogate light chains, we backcrossed mice expressing full-length or truncated μ transgenes with λ5-deficient mice. Our results show that the truncated heavy chain, but not the normal chain, is able to promote pre-B-cell development in the absence of λ5. We also show that truncated μ chains spontaneously aggregate at the surface of bone marrow cells.Conclusion Expression of the truncated μ heavy chain overrides a tightly controlled step of pre-B-cell development, which strongly suggests that a constitutive signal is delivered by the truncated μ chain disease protein. The self-aggregation of μ chain disease proteins might account for this constitutive activation. We conclude that amino-terminal truncation of heavy chains could play a role in the genesis of HCD neoplasia if it occurs at an appropriate stage of B-cell differentiation, namely in a mature B cell

Ins1 Gene Up-Regulated in a β-Cell Line Derived from Ins2 Knockout Mice

The authors have derived a new β-cell line (βIns2−/−lacZ) from Ins2−/− mice that carry the lacZ reporter gene under control of the Ins2 promoter. βIns2−/−lacZ cells stained positively using anti-insulin antibody, expressed β-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 βIns2−/−lacZ cells, as compared to those found in βTC1 cells expressing both Ins1/Ins2. Thus, transcriptional up-regulation of the remaining functional insulin gene in Ins2−/− mice could potentially contribute to the β-cell adaptation exhibited by these mutants, in addition to the increase in β-cell mass that we previously reported.We have also shown that lacZ expression, as analyzed by determining β-galactosidase activity, was up-regulated by incubating βIns2−/−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 activit

Knock-in of diphteria toxin A chain gene at Ins2 locus: effects on islet development and localization of Ins2 expression in the brain.

International audienceWe 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 Ins2(dta/+), Ins2(dta/lacZ) or Ins2(lacZ/+) mouse pups were generated by breeding and analyzed to study the effects of toxigenetic beta-cell ablation on islet development and to localize the extrapancreatic Ins2 expression site in the brain. Ins2(dta/+) and Ins2(dta/lacZ) pups developed a severe diabetic ketoacidosis and died rapidly. Histological analysis of their pancreas revealed that beta-cells completely disappeared in their islets as evidenced by loss of lacZ activity or insulin immunonostaining. beta-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 Ins2(lacZ/+) and Ins2(dta/laZ) 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 beta-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

Human insulin gene expression in transgenic mice: mutational analysis of the regulatory region

International audienc