Construction Of A PiggyBac Transposon(PB) Inducible Cell Immortalization Vector And Its Verification In BMECs

Transposon, a special kind of genomic DNA sequence with mobility, is widely present inbiological genomes as a genetic recombination factor. piggyBac (PB) transposon is a DNAtransposons relied on the function of PB transposase. Numerous studies indicate that PBtransposon can target the tetranucleotide TTAA in the genome of mammalian cells forefficient transposition and is considered the suitable tool in gene tranfer study as a safe andefficient carrier. Human telomerase reverse transcriptase (hTERT),with its own RNAtemplate, maintains and prolongs the length of telomere in most stem cells and cancer cells.The hTERT expression and activation is regarded essential in cell immortalization. In order toconstruct a general cell immortalization vector, we modified the traditional PB transposon,optimized the selectable elements, introduced a hTERT expression cassette and, consequently,increased the integration efficiency and wided the scope of application, which could be atentative exploration on construction of a effective cell immortalization vector. Meanwhile,we transfected the vector constructed into primary bovine mammary epithelial cells (BMECs)in order to obtain cells with hTERT activity, and thus lay the foundation of establishment ofimmortalized bovine mammary epithelial cell lines. The main contents of this study are asfollows:1. We developed an efficient piggyBac (PB) transposon as vector backbone, whichcontained the necessary5’TR and3’TR DNA sequence, two chicken insulators CHS4, a PBtransposase expression cassettes mediated by the TK promoter and a multiple cloning sites(MCS) with10rare restriction sites. Besides, we inserted a selectable element with terminalloxP sequence, which has two maker gene,EGFP and Puro, linked by P2A and a humantelomerase reverse transcriptase(hTERT)expression cassette. We examined and sequenced thevector and tested Cre-loxP system. The results show that the general immortalization vectorpTP-hTERT was successfully constructed. 2. We transfected pTP-hTERT into HEK293cells and screened positive clones. Thepositive cell were examined through RT-PCR, western blotting(WB) and Tail-PCR to verifythe hTERT expression, the functionality of selectable element and flanking sequence of theintegration site in genome,respectively. To confirm the transponson rate,we used methyleneblue stains to observing the cell clones, then counted and analysed the data through unpairedStudent’s t-test. The results showed that the positive cells could be selected by puromycin andhTERT transcript product was detected. The WB results, P2A cutting EGFP and Puro fusionprotein with high efficiency, reflected the selectable elment worked. The Tail-PCR confirmedthat the vector correctly integrated into the cellular genome. The results of methylene bluestaining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERTsignificantly increased(p<0.01) compared with the control group.3. After pTP-hTERT transfection, the monoclonal bovine mammary epithelialcells(BMECs) were obtained, which expressed EGFP and showed the property of anti-puromycin. The hTERT BMECs could express exogenous hTERT protein, from which thetelomerase activity was detected. The preliminary results indicated that pTP-hTERT can beused to establish a immortalized bovine mammary epithelial cell line.