**3.4 Interferon-γ locus regulation with BACs**

To investigate the regulatory properties of conserved non-coding sequence (CNS) element of interferon-γ (Ifng) gene *in vivo*, Hatton et al (Hatton, Harrington et al. 2006) developed a BAC-based transgenic reporter system to express Ifng gene expression. They introduced a Thy1.1 reporter into exon 1 of Ifng and placed this reporter into a BAC containing approximately 60 kb upstream of exon 1 of ifng and approximately 100 kb downstream of exon 4 of ifng sequences. The CNS-22 region of BAC was then flanked with loxP sites (Hatton, Harrington et al. 2006; Wilson and Schoenborn 2006). Because activation of the large BAC transgenic allele unlikely perturbs endogenous alleles (Valjent, Bertran-Gonzalez et al. 2009), potential confounding effects of altered IFNg production were possibly eliminated (Hatton, Harrington et al. 2006). Hatton et al chose Thy1.1 (CD90.1) as a reporter because of its low immunogenicity and easy detection in the context of CD90.2 allotype of the C57BL/6 background. The recombined BACs (Ifng-Thy1.1 BAC) containing the Thy1.1 reporter and floxed CNS-22 were microinjected into fertilized C57BL/6 oocytes (Hatton, Harrington et al. 2006; Wilson and Schoenborn 2006). As a result, the Ifng-Thy1.1 BAC-in transgene completely mirrored endogenous Ifng gene expression; and conditional deletion of the CNS-22 element from the single copy transgene by Cre recombinase resulted in almost complete loss of Thy1.1 expression in Th1 cells, CD8+ T cells, and NK cells irrespective of activation through the T cell receptor (TCR)-dependent or TCR-independent pathways. Thus, CNS-22 is considered to be critically involved in Ifng gene expression, irrespective of adaptive or innate immune cell lineage (Hatton, Harrington et al. 2006; Wilson and Schoenborn 2006). CNS-22 functions as an enhancer both *in vitro* and *in vivo*, which will shed new light on ifng regulation and open up avenues for future investigation.

### **3.5 Studies of Kras-mediated pancreatic tumorigenesis with BACs**

Activation of Kras gene by mutation plays a critical role in human pancreatic cancer (Almoguera, Shibata et al. 1988; Shibata, Almoguera et al. 1990). Although the known capability of oncogenic Kras to function as a key initiator of pancreatic malignancy, the mechanism(s) of Kras-caused initiating events are still unclear. This is an important reason why prognosis for patients with malignant pancreatic tumors have not entirely improved over the past twenty years (Jemal, Siegel et al. 2006). Moore et al have discovered that the zebrafish develop pancreatic cancer after exposure to chemical mutagens (Moore, Rush et al. 2006). The studies have also shown that mammalian and zebrafish pancreas are significantly similar in anatomy and histology (Wallace and Pack 2003; Chen, Li et al. 2007). Therefore, the Zebrafish has emerged as an experimental model for study of human pancreatic cancer biology (Davison, Woo Park et al. 2008; Park, Davison et al. 2008). Another benefits of working with zebrafish model is their translucency, which greatly improves the visualization of fluorescent trangenes in both embryos and adult zebrafish (Davison, Woo Park et al. 2008). Park et al (Park, Davison et al. 2008) discovered that oncogenic Kras causes pancreatic cell expansion and malignant transformation in the zebrafish exocrine pancreas by utilizing eGFP-Kras BAC transgenes (160kb) under the regulation of Ptf1a regulatory elements. Ptf1a induces differentiation, growth and proliferation of pancreatic progenitor cells (Park, Davison et al. 2008). Briefly, they expressed either extended green fluorescent protein (eGFP) alone or eGFP fused to oncogenic Kras in developing zebrafish pancreas and continuously detected the expression of fluorescent transgenes transcutaneously during all stages of development including the adult zebrafish. They first generated polymerase chain reaction (PCR) products encoding the eGFP and eGFP-Kras transgenes flanked by sequences homologous to the CH211-142 BAC that spans the Pfta1 gene locus. Homologous recombination leads to accurate replacement of the Ptf1a coding sequences with the eGFP and eGFP-Kras transgene (Davison, Woo Park et al. 2008). Their results demonstrate that oncogenic Kras-expressed pancreatic progenitor cells fail to undergo characteristic exocrine differentiation although their initial specification and migration are observed to be normal (Davison, Woo Park et al. 2008; Park, Davison et al. 2008). Blocks of differentiation leads to abnormal accumulation of the undifferentiated progenitor cells, correlates with the formation of invasive pancreatic cancer. Besides similarity in anatomy and histology, Zebrafish pancreatic tumors share several activated signaling pathways with the human pancreatic tumors, including activation of ERK and AKTby phosphorylation, as well as abnormal Hedgehog pathway activation which was justified by the up-regulation of ptc1 mRNA and gli1 mRNA (Park, Davison et al. 2008). These findings provide a unique view of the tumor-initiating effects of oncogenic Kras in a living vertebrate organism, but more important it suggest that BACs transgene targeting other oncogenes or tumor suppressor genes in zebrafish pancreatic cancer may improve our understanding of the human disease.

#### **3.6 Studies on striatal signaling pathways in central nervous system (CNS) with BACs**

To understand the role of molecular signaling pathways involved in behavioral responses, it is necessary to delineate the molecular events that take place in neurons. This task has been hampered by the complexity of neuronal system. There are hundreds of distinct neuronal populations and these populations are very difficult to distinguish (Valjent, Bertran-Gonzalez et al. 2009). The development of BAC transgenic mice expressing various reporters, epitope tagged-proteins or Cre recombinase driven by specific promoters, greatly facilitates the research in this field. Generally speaking, transgene expression is influenced by copy numbers and site of insertions (positional effects). Large BAC transgenes (BACs contain large fragments 150-200kb of mouse genome) have usually a low copy number, are less likely influenced by positional effects, and are able to recapitulate the regulation of endogenous genes much better than shorter transgenes (Yang, Model et al. 1997). Over the past few years, the use of BAC EGFP transgenic mice have generated significant development in the analysis of striatal physiology and physiopathology (Valjent, Bertran-Gonzalez et al. 2009). The drd1a-EGFP (EGFP reporter is driven by dopamine D1 receptor-D1R promoter), drd2-EGFP (EGFP reporter is driven by dopamine D2 receptor-D2R promoter) and chrm4-EGFP (EGFP reporter is driven by cholinergic receptor, muscarinic 4- CHRM4 receptor promoter) BAC transgenic mice have been extensively utilized to investigate the physiological features of striatonigral and striatopallidal medium spiny projection neurons (MSNs) (Lobo, Karsten et al. 2006; Kreitzer and Malenka 2007; Cepeda, Andre et al. 2008; Gertler, Chan et al. 2008). Among these major findings are as follows: 1)D1R-expressing MSNs are less excitable than D2R–MSNs (Lobo, Karsten et al. 2006; Kreitzer and Malenka 2007; Cepeda, Andre et al. 2008; Gertler, Chan et al. 2008) due to different morphology (Gertler, Chan et al. 2008), and some presynaptic factors (Kreitzer and Malenka 2007; Cepeda, Andre et al. 2008). Corticostriatal synapses are activated by repetitive stimulation; in contrast, thalamostriatal synapses are inhibited by repetitive stimulation (Ding, Peterson et al. 2008); 2) D1R-expressing MSNs collaterals are functionally connected primarily with other D1R–MSNs, whereas D2R-expressing neurons collaterals are connected with both D2R– and D1R–MSNs (Taverna, Ilijic et al. 2008). D2R–MSNs synapse with GABAA receptors are stronger (Taverna, Ilijic et al. 2008) and generate greater GABAA receptor-mediated tonic currents (Ade, Janssen et al. 2008) than D1R–MSNs (Janssen, Ade et al. 2009); 3)The single back-propagating action potentials invade more distal dendritic regions in D2R–than in D1R–MSNs, due to a difference in voltage-dependent Na+channels and Kv4 K+ channels (Day, Wokosin et al. 2008); 4) In the dopamine-depleted striatum, the corticostriatal connections are decreased in D2R neurons (Day, Wang et al. 2006), whereas dendritic excitability is increased in this region (Day, Wokosin et al. 2008; Taverna, Ilijic et al. 2008).
