The Preliminary Study Of BCR-ABL Fusion Gene Transgenic Mice With Tet-On System

Chronic myelogenous leukemia (CML) is a clonal disorder originating in the hematopoietic stem cell shown to be associated with a specific chimeric fusion gene-BCR/ABL. In order to study the mechanism of CML, many transgenic mice models have been developed. But most of BCR-ABL transgenic mice models cannot resemble the natural course of human CML.To control the level and timing of P210bcr-abl expression in an adult mouse, a tetracycline regulable tet-on expression system has been employed. The regulation of the reverse tetracycline-controlled transactivator (rtTA) is controlled by bcr promoter, which shows relative specificity in mice hematopoietic stem cells and myeloid cells. We employed tet-on inducible expression system to rescue the early lethality of P210bcr-abl. This model is expected to effectively develop a CML-like disease, which will provide a good tool for the study of pathogenesis of CML, biology of leukemic stem cell and developing new therapeutic reagents.In preliminary study, we had successfully cloned a 1.1 Kb human bcr promoter and validated its relative specificity in myeloid cells. We have constructed the recombinant expression vector pbcr-Tet-On-tTs and pTRE2-BCR-ABL-Hyg. Then we co-transfectd HL-60 cells with two vectors of pbcr-Tet-On-tTs and pTRE2-BCR-ABL-Hyg, and induced the expression of rtTA with doxycycline. The result showed the expression of P210bcr-abl was positive proportion to the concentration of doxycycline. Transgenic mice were established by microinjection of bcr-tet-On-tTS and TRE2-BCR-ABL genes into male nuclei of zygote from ICR×C57BL/6 F1 mice.It is an important part to screen out transgenic animals of transgenic animal preparation. Because of its high sensitivity and simplicity, polymerase chain reaction (PCR) becomes a conventional method to detect transgenic animals. In order to improve the accuracy and credibility of PCR method, we optimized the PCR reaction from many aspects, such as the template quality, primers, Taq enzyme concentration, Mg2+ concentration and annealing temperature. At the same time we followed quality assurance/quality control (QA/QC) procedures carefully, collaborative with ultraviolet radiation to prevent environmental DNA contamination, to reduce the chance of false-positive results.In this study, we transplanted 2550 microinjected zygotes into 116 receptors mice and got 212 transgenic founder mice. The success rate of microinjection was 60.9%, the mice birth rate was 8.3%. TRE2-BCR-ABL and bcr-tet-On-tTS double injection of gene transfer eggs 1709, was born 37 mice, mice birth rate was 2.2 percent; TRE2-BCR-ABL gene-transfer eggs injected 281, born 25 mice, mice birth rate was 8.9 percent; bcr-tet-On-tTS single injection of gene transfer 560 eggs, born 150 mice, mice birth rate was 26.8 percent. We screening the born mice with optimize PCR method, three mouse of positive TRE2-BCR-ABL were found, one of them were confirmed by Southern blot detetion. It is a pity for we didn't detect positive of TRE2-BCR-ABL in it's all of offspring. At present, we had not get TRE2-BCR-ABL and bcr-tet-On-tTS positive mouse.At present, we are actively analyzing the cause, expect as soon as possible to get TRE2-BCR-ABL and bcr-Tet-On-tTS transgenic mice models.