AbstractsBiology & Animal Science

PiggyBac transposon mutagenesis in human and mouse stem cells

by Tingting Zhang

Institution: University of Alabama – Birmingham
Degree: PhD
Year: 2011
Keywords: DNA Transposable Elements – genetics.<br> Hemoglobins – genetics<br> Mutagenesis, Insertional – genetics<br>PGBD1 protein, human
Record ID: 1910133
Full text PDF: http://contentdm.mhsl.uab.edu/u?/etd,1358


The most common genetic diseases worldwide, ? thalassemia and Sickle Cell Anemia (SCA) result from mutations in the adult ? globin gene. One promising therapeutic method to ameliorate these diseases is to reactivate the endogenous fetal ? globin gene. I hypothesize that an erythroid mutagenic transposon system can be used to activate ? globin gene expression. To test this hypothesis three lines of genetically modified mice were generated and bred together. The first is a transgenic mouse line that contains a multicopy array of erythroid mutagenic PiggyBac (PB) transposons. The second transgenic mouse line expresses the PB transposase (TPase) from a GATA1 enhancer/promoter specifically in erythroid tissues. The final mouse produced by gene targeting in embryonic stem (ES) cells has the murine adult ? globin genes replaced by a human fetal to adult hemoglobin switching cassette. In this mouse the knockin human fetal ? globin promoter drives expression of EGFP during fetal life before switching to a non-functional human ? globin allele. In double transgenic mice I demonstrate the hematopoietic specific expression of PiggyBac transposase can excise the erythroid mutagenic transposon. In addition, individual subclone of transposon-transduced ES cells was established. The chromosomal integration site of each transposon was precisely mapped in these ES cell lines. Mutant mice that were 100% ES cell derived were generated by microinjection of transposon-transduced ES cells into eight-cell embryos. Mice produced from ES cells with multiple transposon integrations were backcrossed to control mice to generate animals with single transposon insertions. Heterozygous mice with PB transposon integration into an exon of the glucosamine phosphate deaminase 1 (GNPDA1) gene were bred to generate homozygotes. Homozygosity of the GNPDA1 insertional mutation resulted in embryonic death around 6.5 days of development. ES cell lines were generated from homozygous GNPDA1 mutant blastocysts. GNPDA1 deficient ES cells could still differentiate into embryoid bodies in vitro and form teratomas in vivo. After pro-apoptotic stimulation GNPDA1 deficient ES cells had significantly increased apoptosis over control ES cells. GNPDA1 deficient ES cells exhibited changes in O-linked N-acetylglucosamine (O-GlcNAc) modifications of cytosolic proteins under no/low glucose or high glucosamine cell culture conditions. 1 online resource (xiv, 128 p.) :ill., digital, PDF file. Biochemistry And Molecular Genetics Joint Health Sciences hemoglobin switching, PiggyBac transposon, ES cells, insertional mutagenesis, GNPDA1 UNRESTRICTED