Wen K. Yang

Thymus Involution Induced by 5-Azacytidine in the Mouse is by Selected Depletion of Immature Thymocyte Subsets

5-Azacytidine, a cytidine analogue well known for its inhibitory effect on DNA methylation of mammalian cells, has been used for chemotherapy of cancers, particularly childhood leukemias, but is also known for its ability to cause thymus involution and lymphoma induction in animal experiments. To understand the acute cytotoxic effects on the thymus, young laboratory mice treated with sublethal doses of 5-azacytidine were examined by immunofluorescent cytometric measurements of thymocyte populations and also by histological and DNA analyses of cell death characteristics. Young FVB/N strain mice, which were injected with 5-azacytidine every other day for three times and examined 48 hours later, showed marked reduction in thymus weight and cellularity. This was primarily due to virtual elimination of the CD4+CD8+ doubly positive cell populations from the thymus. Kinetic changes following a single injection of 5-azacytidine indicated that the thymocytes most susceptible to depletion by 5-azacytidine were those thymocyte subsets that carry low to undetectable levels of the CD3 antigen of the T cell receptor complex and hence represent the precursor and presumably the apoptosis-prone subsets of the CD4+CD8+ population. However, in contrast to the histological feature and nucleosomal DNA fragmentation characteristic of apoptosis observed in the dexamethasonetreated mice, early pyknotic changes of htymocytes in thymic cortex and diffuse chromatin DNA degradation were observed in 5-azacytidine-treated mice. These results suggest that acute cytotoxicity of 5-azacytidine involves necrotic changes of the immature CD4+CD8+ thymocyte subsets in the thymus.

Reproductive, Genetic and Oncogenic Complications in Transgenic Mice Developed by Micro-injection of Recombinant DNA Constructs That Contain Mouse Chromosomal Retroviral Long-Terminal-Repeat Elements

Six different recombinant DNA clones, each containing a marker gene that carries at its 5’ side the long-terminal-repeat (LTR) sequence of endogenous ecotropic murine leukemia virus or two related mouse chromosomal retroviral gene families, were micro-injected into fertilized mouse eggs of FVB/N inbred strain for the purpose of generating transgenic mice. After overnight incubation, 1373 of the 3154 micro-injected fertilized eggs divided into two-cell embryos which were transferred, 10 to 15 per oviduct, into pseudo-pregnant B6C3Fl mice. From a total of 1297 two-cell embryos transferred, about one fourth developed into mature fetuses in the surrogate mother uteri and were born; but after birth many neonates were lost due to maternal cannibalism. Only 168 live FVB/N weanlings were obtained and 34 of them were tested positive of the micro-injected endogenous retroviral LTR transgenes by dot-blot analysis of the tail DNA preparation. The apparent failure of DNA micro-injected zygote to undergo the first cell division and subsequent embryo development did not differ significantly among the 6 LTR-marker gene constructs used for microinjection, suggesting the transgenic procedures or processes as the major cause of abnormal embryo development. From 5-6 weeks of age, each of these 34 original generation transgenic mice was individually housed with fertile non-transgenic FVB/N mice of the opposite sex for mating. Poor reproductive performance was found in six, including three completely sterile and runted. Suggesting dominant genetic defects in reproductive performance. The remaining fertile transgenic mice could serve as “founders” to generate F1 and subsequent generations of homozygous progeny, leading to distinct transgenic mouse lines in FVB/N genetic background. Examination of the progeny mice generated from fertile founders showed that the maker gene expression was usually undetectable and the LTR portion of the transgene-DNA was methylated in various organs, suggesting a silent state of transcription of the LTR-containing transgenes. An obvious recessive mutation affecting neuro-muscular function was observed. Other than apparent transgene instability in some of female founders and apparent leukemogenic effects of a LTR interleukin-3 marker gene, as previously reported, increased tumor incidence was observed in some founder mice and their descendent generations. In the female progeny of a transgenic mouse line that carried a mouse Y-chromosome (male-specific) LTR elements, there was an apparent increase of tumor incidence in the reproductive organs, such as ovary, uterus and mammary gland.