Browsing by Subject "Gene targeting"
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Item Acyl CoA Binding Protein (ACBP) Gene Ablation Induces Pre-Implantation Embryonic Lethality in Mice(2012-02-14) Landrock, DaniloUnique among the intracellular lipid binding proteins, acyl CoA binding protein (ACBP) exclusively binds long chain fatty acyl CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not result in any live homozygous deficient (null) ACBP^(-/-) pups. Heterozygous and wild type embryos were detected at all postimplantation stages, but no homozygous ACBP null embryos were obtained? suggesting that an embryonic lethality occurred at a preimplantation stage of development, or that embryos never formed. While ACBP null embryos were not detected at any blastocyst stage, ACBP null embryos were detected at the morula (8- cell), cleavage (2-cell), and zygote (1-cell) preimplantation stages. Two other LCFACoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCPx) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins only at the earliest preimplantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.Item Bacterial gene targeting using group II intron L1.LtrB splicing and retrohoming(2008-05) Yao, Jun, 1974-; Lambowitz, AlanThe Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base pairing of the intron RNA. Consequently, Ll.LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll.LtrB introns contains most viable E. coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. I also demonstrate that Ll.LtrB introns can be used for efficient gene targeting in a variety of Gram-negative and positive bacteria, including E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Bacillus subtilis, and Staphylococcus aureus. Ll.LtrB introns expressed from a broad-host-range vector or an E. coli-S. aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1-100% without selection. By using an Ll.LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, I disrupted the essential gene hsa in S. aureus. Because the splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32oC, but not at 43oC. Finally, I developed high-throughput screens to identify E. coli genes that affect either the splicing or retrohoming of the Ll.LtrB intron. By using these screens, I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies, in some cases apparently resulting from increased stability of the intron RNA.Item DNA target site recognition and toward gene targeting in mammalian cells by the Ll.LtrB group II intron RNP(2013-05) Hanson, Joseph Haskell; Lambowitz, AlanMobile group II introns insert site-specifically into DNA target sites through a mechanism ("retrohoming") that involves reverse splicing of the intron RNA into the DNA and its subsequent reverse transcription by an intron-encoded protein (IEP) that is associated with the RNA in a ribonucleoprotein (RNP) complex. Characterization of this RNP complex and its retrohoming activities have enabled the development of programmable mobile group II intron gene targeting vectors routinely used in prokaryotic organisms. Building upon recent research by our lab to develop gene targeting in Xenopus laevis and Drosophila melanogaster using the group II intron Ll.LtrB from Lactococcus lactis, I describe work to extend this system to mammalian cells. I demonstrate that group II intron RNPs can be delivered to mammalian cells efficiently and produced in vivo via a CMV/T7 hybrid expression system. Using a robust single-strand annealing assay to detect homologous recombination induced by double-strand breaks (DSBs), I found that group II intron-mediated DSBs are efficiently repaired by mammalian cells. Despite varied approaches, I failed to detect endogenous group II intron-mediated gene targeting in human and mouse cells in culture. Gene expression microarray analysis and in vivo imaging of RNP molecules indicated that group II intron RNPs are sequestered away from the genome and induce host innate immune responses. I also investigated how the C-terminal DNA-binding domain of the Ll.LtrB IEP contributes to DNA target site recognition. Building upon previous mass spectrophotometric analysis of site-specific UV-crosslinking, I used genetic and biochemical analyses to identify potential protein contacts for key target site residues T-23 and T+5. Genetic selection of mutants in a region contacting T+5 led to identification of LtrA variants with increased retrohoming efficiency. My results provide evidence that the DNA-binding domain of a group II intron reverse transcriptase functions in DNA target site recognition and suggest new methods for changing its DNA target specificity and targeting efficiency.Item Function and regulation of CCAAT/enhancer binding protein beta in Leydig cell development and steroidogenesis(Texas Tech University, 2002-08) Nalbant, DemetPituitary luteinizing hormone (LH) is required for the development of multiple cell types in both the male and female reproductive systems. In particular, LH promotes differentiation of testicular Leydig cells and stimulates steroid production by Leydig cells in the testis, theca, granulosa and luteal cells in the ovary. We have been interested in identifying nuclear transcription factors that are targets of LH signaling pathways in Leydig cells and concentrated on CCAAT/enhancer binding protein beta (C/EBPp). Our initial studies showed that C/EBPp is expressed in a differentiation specific pattern whose expression is stimulated by LH/human chorionic gonadotropin (hCG) and cyclic AMP (cAMP) in Leydig cells. We hypothesized that C/EBPp plays an important role in LH regulated Leydig cell development and steroidogenic function. To assess the specific roles of C/EBPp in Leydig cell function we have analyzed the steroidogenic capacity of Leydig cells from C/EBPp-deficient mice generated by gene targeting. This study revealed that testosterone production in male C/EBPp deficient mice is severally compromised suggesting that C/EBPp is essential for complete functional differentiation of Leydig cells. In order to understand how LH effects on Leydig cell differentiation and/or function may be mediated through C/EBPp, we attempted to identify genetic control elements that control C/EBPp transcription in steroidogenic and non-steroidogenic cells. We identified an evolutionarily conserved, steroidogenic cell-specific, distal enhancer element located in the C/EBPp 5'-flanking region. Our studies suggest that the activity of the enhancer may be, at least in part, controlled by as yet uncharacterized nuclear factors specifically detected in nuclear extracts of steroidogenic cells. These studies set the stage for elucidation of the molecular mechanisms controlling C/EBPp expression in steroidogenic cells in general, and may aid in uncovering alternative LH-dependent signaling pathways critical for functional maturation of Leydig cellsItem Group II intron and gene targeting reactions in Drosophila melanogaster(2011-08) White, Travis Brandon; Lambowitz, Alan; Bull, James J.; Macdonald, Paul M.; Paull, Tanya T.; Stevens, Scott W.Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms.Item Group II intron mobility and its application in gene targeting(2003-08) Zhong, Jin, 1972-; Lambowitz, AlanItem Toward group II intron-based genome targeting in eukaryotic cells(2009-12) Vernon, Jamie Lee; Lambowitz, AlanMobile group II introns consist of a self-splicing RNA molecule and an intron-encoded protein with reverse transcriptase activity that function together in an RNP and catalyze the insertion of the intron into specific DNA target sites by a process known as retrohoming. The mechanism of insertion requires the intron RNA to bind and reverse splice into one strand of the DNA target site, while the intron-associated protein cleaves the opposite DNA strand and reverse transcribes the intron RNA. DNA target site recognition and binding are dependent upon base pairing between the intron RNA and the target DNA molecule. By modifying the recognition sequences in the intron RNA, group II introns can be engineered to insert into virtually any desired target DNA. Based on this technology, a novel class of commercially available group II intron-based gene targeting vectors, called targetrons, has been developed. Targetrons have been used successfully for gene targeting in a broad range of bacteria. Previously, our laboratory demonstrated that group II introns retain controllable retrohoming activity in mammalian cells, albeit with very low targeting efficiency. However, the gene targeting capability of group II introns is not limited to direct insertion of the intron. Group II introns can also create double-strand breaks that stimulate homologous recombination. By virtue of these attributes, mobile group II introns offer great promise for applications in genetic engineering, functional genomics and gene therapy. Here I present the results of experiments in which I tested group II introns for gene targeting activities in eukaryotic cells. First, I demonstrated that group II introns injected into zebrafish (Danio rerio) embryos retain in vivo plasmid targeting activity that is enhanced by the addition of magnesium chloride and deoxynucleotides. I also verified that similar in vivo targeting activity is retained in Drosophila melanogaster embryos. Further, I describe repeated experiments in zebrafish embryos designed to target the zebrafish genome with inconclusive results. Group II introns were also delivered to cultured human cells for genome targeting. Here I present promising evidence for the ability of group II introns to stimulate homologous recombination between an exogenously introduced donor DNA molecule and the chromosome. The donor DNA was delivered either as a linearized double-stranded plasmid by electroporation or as a single stranded genome of a recombinant adeno-associated virus (AAV). In both cases, cells receiving both the group II intron RNP and the donor DNA showed more efficient integration of the donor DNA than introduction of the donor DNA alone. The studies presented here provide insight into the potential of using group II introns for future applications in gene targeting in eukaryotes.