Construction Of Subtractive CDNA Library Of Familial Acute Myelogenous Leukemia And Cloning,Evaluation And Sequence Analysis Of Differential Expressed Genes

Acute leukemia is a common malignant tumour of hematological system. It hasbeen proved by former researches that its pathogenesis relates to multi-genes andmulti-stages. However, it is still not enough evidence to explain it by current researchdata. So, isolating biological active substances in acute leukemia processes andrevealing their functional approaches are very important to understand molecularmechanisms of acute leukemia. The heredity families provide us the better samplesthat are helpful to understand the related virulence genes. 【Objective】To isolategenes expressed differentially from bone marrow cells of familial acute myelgenousleukemia and to explain the molecular mechanisms of the disease at the gene level.【Methods 】Super SMART cDNA synthesis and suppression subtractivehybridization (SSH) were performed to isolate differentially expressed cDNAfragments from patient's bone marrow cells. cDNA from the patient were used as"tester", the other from the normal human as "driver". Subtractive products weredirectly inserted into T/A cloning vector, and then transformed into host bacteria, toset up a subtractive cDNA library of specially or highly expressed genes in familialacute myelogenous leukemia. Those clones were screened and identified by reformeddifferential screening technique which the probes were labeled with DIG. Positiveclones were sequenced and compared with known sequences in the public databasesof GenBank/EMBL/DDBJ using NCBI BLAST for homology analysis. The unknownfragments were then submitted to GenBank. 【Results】Total RNA were extractedand purified in good quality. Double strand cDNA were reverse transcriptedintegratedly, and then cut by RsaⅠinto even length short segments. Ligation wasidentified highly effective. After two hybridizations, a subtractive library ofdifferentially expressed genes in human familial acute myelgenous leukemia wasconstructed successfully by SSH. Nested-PCR was specificial, which amplified thedifferentially expressed genes exponentially. Subtractive products were inserted intothe T/A clone vectors and then transformed into TOPO10F'competent cellssuccessfully, which dispersed differentially expressed genes of the library into a singlecopy. After differential screening and RT-PCR, 28 clones were confirmed to bepositive differentially expressed genes fragments. Sequences were submitted toGenBank for homology analysis to determine what kind of protein they were encoding,and confirm which fragments were new ESTs. Then 17 sequences were a part ofknown genes, and the rests were verified unknown fragments.【Conclusion】(1) SSHis an effective approach to isolate differentially expressed genes. (2) Labeling probeswith DIG was applied in differential screening technology which substituted for theconventional P32-dCTP isotope probes. This technique was not only maintaining thehigh sensitivity and specificity but also averting isotope pollution and simplifyingexperimental procedures. (3) Some genes related to cell proliferation anddifferentiation were acquired by SSH, which proved sufficiently that the occurrence ofacute leukemia was involved in multi-genes and multi-stages. It is a complex process.(4) Eleven novel ESTs related to library were successfully isolated and accepted byGenBank, the access numbers are CX129926-7, CV884199, CV884202-3,CV973096-101, respectively.