BAC Transgenesis Research Articles

Transgenic Tg(Kcnj10-ZsGreen) fluorescent reporter mice allow visualization of intermediate cells in the stria vascularis

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen’s, Deiters’ and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP. These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Loss of mouse Stmn2 function causes motor neuropathy

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration accompanied by aberrant accumulation and loss of function of the RNA-binding protein TDP43. Thus far, it remains unresolved to what extent TDP43 loss of function directly contributes to motor system dysfunction. Here, we employed gene editing to find whether the mouse ortholog of the TDP43-regulated gene STMN2 has an important function in maintaining the motor system. Both mosaic founders and homozygous loss-of-function Stmn2 mice exhibited neuromuscular junction denervation and fragmentation, resulting in muscle atrophy and impaired motor behavior, accompanied by an imbalance in neuronal microtubule dynamics in the spinal cord. The introduction of human STMN2 through BAC transgenesis was sufficient to rescue the motor phenotypes observed in Stmn2 mutant mice. Collectively, our results demonstrate that disrupting the ortholog of a single TDP43-regulated RNA is sufficient to cause substantial motor dysfunction, indicating that disruption of TDP43 function is likely a contributor to ALS.

Of mice and human-specific long noncoding RNAs.

The number of human LncRNAs has now exceeded all known protein-coding genes. Most studies of human LncRNAs have been conducted in cell culture systems where various mechanisms of action have been worked out. On the other hand, efforts to elucidate the function of human LncRNAs in an in vivo setting have been limited. In this brief review, we highlight some strengths and weaknesses of studying human LncRNAs in the mouse. Special consideration is given to bacterial artificial chromosome transgenesis and genome editing. The integration of these technical innovations offers an unprecedented opportunity to complement and extend the expansive literature of cell culture models for the study of human LncRNAs. Two different examples of how BAC transgenesis and genome editing can be leveraged to gain insight into human LncRNA regulation and function in mice are presented: the random integration of a vascular cell-enriched LncRNA and a targeted approach for a new LncRNA immediately upstream of the ACE2 gene, which encodes the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent underlying the coronavirus disease-19 (COVID-19) pandemic.

An inducible Cre mouse line to sparsely target nervous system cells, including Remak Schwann cells

Nerves of the peripheral nervous system contain two classes of Schwann cells: myelinating Schwann cells that ensheath large caliber axons and generate the myelin sheath, and Remak Schwann cells that surround smaller axons and do not myelinate. While tools exist for genetic targeting of Schwann cell precursors and myelinating Schwann cells, such reagents have been challenging to generate specifically for the Remak population, in part because many of the genes that mark this population in maturity are also robustly expressed in Schwann cell precursors. To circumvent this challenge, we utilized BAC transgenesis to generate a mouse line expressing a tamoxifen-inducible Cre under the control of a Remak-expressed gene promoter (Egr1). However, as Egr1 is also an activity dependent gene expressed by some neurons, we flanked this Cre by flippase (Flpe) recognition sites, and coinjected a BAC expressing Flpe under control of a pan-neuronal Snap25 promoter to excise the Cre transgene from these neuronal cells. Genotyping and inheritance demonstrate that the two BACs co-integrated into a single locus, facilitating maintenance of the line. Anatomical studies following a cross to a reporter line show sparse tamoxifen-dependent recombination in Remak Schwann cells within the mature sciatic nerve. However, depletion of neuronal Cre activity by Flpe is partial, with some neurons and astrocytes also showing evidence of Cre reporter activity in the central nervous system. Thus, this mouse line will be useful in mosaic loss-of-function studies, lineage tracing studies following injury, live cell imaging studies, or other experiments benefiting from sparse labeling.

Cux2 expression regulated by Lhx2 in the upper layer neurons of the developing cortex

The laminar structure, a unique feature of the mammalian cerebrum, is formed by a number of genes in a highly complex process. The pyramidal neurons that make up each layer of the cerebrum are functionally characterized by specific gene expressions. In particular, Cux1 and Cux2, which are specifically expressed in layer II-IV neurons, are known to regulate dendritic branching, spine morphology, and synapse formation. However, it is still unknown how their expression is regulated transcriptionally. Here we constructed Cux2-mCherry transgenic mice that reproduce the cortical layer II-IV-specific expression of Cux2, a member of the Cut/Cux/CDP family, using BAC transgenesis and a variety of coordinated cortical layer markers that are known to date. Our immunohistochemistry analysis shows that mCherry was expressed in cortical layer II-IV and the corpus callosum in the same way as endogenous Cux2 without ectopic expression. We also identified a region of 220 bp that is highly conserved in mammals and controls specific cerebral expression of Cux2, using comparative genome analysis and in vivo reporter assays. Furthermore, we confirm that Lhx2, whose expression in cortical layer II-IV is similar to that of the Cux2 enhancer, can act as a transcriptional activator. These results suggest that cortical layer II-IV expression of Cux2 can be regulated by the interaction of Cux2-E1 and Lhx2, and that their failure to co-regulate is associated with neurodevelopmental disorders such as autism and schizophrenia.

Testis-specific Arf promoter expression in a transposase-aided BAC transgenic mouse model.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFβ-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

The Cdkn1aSUPER Mouse as a Tool to Study p53-Mediated Tumor Suppression

Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-drivenlung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression.

PD147: Characterization of the newly identified periodontitis‐associated gene Spag4 using a physiologically regulated reporter cell line generated by BAC Transgenesis

PD147: Characterization of the newly identified periodontitis‐associated gene Spag4 using a physiologically regulated reporter cell line generated by BAC Transgenesis

Novel hypophysiotropic AgRP2 neurons and pineal cells revealed by BAC transgenesis in zebrafish

The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate.

No privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats.

BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.

Abstract 4180: Rewiring the cytokine network in melanoma

Abstract Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Fortuitously, malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. T helper type-1 lymphocytes (TH1) play a pivotal role in inducing and maintaining anti-tumour immune responses. In melanoma patients, it has been observed that higher number of TH1 cells in the tumour micro-environment lead to better prognosis. In this study, using an autochthonous zebrafish melanoma model, when we forced the expression of interferon gamma (IFNg) locally in the tumour microenvironment, thereby potentially enhancing TH1 differentiation, we observed an inflammatory response against melanoma that eventually lead to complete tumour regression. Using transposon mediated BAC transgenesis, we have generated a Tg(cd4:mcherry) reporter line labelling CD4+ cells in zebrafish. By driving Human NRASQ61L in zebrafish melanocytes we have modelled melanoma in zebrafish and later when these fish developed tumours we forced the expression of IFNg in melanoma cells using a tamoxifen inducible LSL/CreERT2 system driven by mitfa minimal promoter. Experiments were performed using animals with and without CreERT2. Following tamoxifen administration, we observed a loss of pigmentation in all the IFNg induced tumours (n = 12) by 2 weeks post induction which gradually increased over the study period. Whereas no loss of pigmentation was observed in control tumours (n = 12) throughout the study period. At 9 weeks post induction, the study was terminated and tumours were analysed using histology. In tumours were IFNg was induced, we observed that the tumour tissue was now replaced with fibrotic tissue accompanied by a marked lymphocyte infiltration and patches of necrosis. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. We observed no signs of tumour regression in control tumours. Although preliminary, our data is very exciting and promising as it will open new avenues for developing combinatorial therapies in melanoma. Using a high throughput in-vivo model system such as zebrafish, it is an exciting proposition to test the impact of combining anti-PD1/PDL1 inhibitors with various cytokines/chemokines in inducing effective and sustained tumour regression in melanoma. Citation Format: Raghavendar Nagaraju, Christopher Dee, Helen Young, Jorge Barriuso, Christopher Secombes, Adam Hurlstone. Rewiring the cytokine network in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4180.

A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types.

There is a continuing need for driver strains to enable cell-type-specific manipulation in the nervous system. Each cell type expresses a unique set of genes, and recapitulating expression of marker genes by BAC transgenesis or knock-in has generated useful transgenic mouse lines. However, since genes are often expressed in many cell types, many of these lines have relatively broad expression patterns. We report an alternative transgenic approach capturing distal enhancers for more focused expression. We identified an enhancer trap probe often producing restricted reporter expression and developed efficient enhancer trap screening with the PiggyBac transposon. We established more than 200 lines and found many lines that label small subsets of neurons in brain substructures, including known and novel cell types. Images and other information about each line are available online (enhancertrap.bio.brandeis.edu).

BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene.

SummarySingle Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity‐related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk‐associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter‐GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in‐frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk‐associated region. genesis 53:640–651, 2015. © 2015 The Authors. genesis Published by Wiley Periodicals, Inc.

Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons.

Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3'-Untranslated Region Polymorphism C+87T by MicroRNA-107.

Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3'-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3'-untranslated region (3'-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3'-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3'-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

In vitro human embryonic stem cell hematopoiesis mimics MYB independent yolk sac hematopoiesis

The in vitro formation of HSC from hESC has been attempted by many scientists because of its clinical applicability. Here we investigated whether human in vitro hematopoiesis generates cMYB dependent HSC (precursors) besides HSC independent yolk sac (YS) type precursors. We generated cMYBeGFP reporter hESC using random integration BAC transgenesis and could show that GFP expression paralleled cMYB expression. Upon hematopoietic differentiation, eGFP weakly positive cells became first apparent from d11 onwards, forming a clear population by d14.

Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond

The development and application of diverse BAC transgenic rodent lines has enabled rapid progress for precise molecular targeting of genetically-defined cell types in the mammalian central nervous system. These transgenic tools have played a central role in the optogenetic revolution in neuroscience. Indeed, an overwhelming proportion of studies in this field have made use of BAC transgenic Cre driver lines to achieve targeted expression of optogenetic probes in the brain. In addition, several BAC transgenic mouse lines have been established for direct cell-type specific expression of Channelrhodopsin-2 (ChR2). While the benefits of these new tools largely outweigh any accompanying challenges, many available BAC transgenic lines may suffer from confounds due in part to increased gene dosage of one or more “extra” genes contained within the large BAC DNA sequences. Here we discuss this under-appreciated issue and propose strategies for developing the next generation of BAC transgenic lines that are devoid of extra genes. Furthermore, we provide evidence that these strategies are simple, reproducible, and do not disrupt the intended cell-type specific transgene expression patterns for several distinct BAC clones. These strategies may be widely implemented for improved BAC transgenesis across diverse disciplines.

Regulation of chromogranin A by hsa‐miR‐107 in hypertension and nephropathy (1136.19)

Chromogranin A (CHGA) is a major protein in secretory vesicles of neuroendocrine cells, co‐released with catecholamines. Genetic variation in the CHGA 3’‐UTR region is associated with hypertensive nephropathy. This study documents the biological mechanism underlying the genetic association of marker and trait.Methods and Results: An African‐American population case/control study showed that individuals carrying the CHGA 3’‐UTR variant C+87T (rs7610) haplotype are likely to develop end stage renal disease. Computational analysis showed that the variant is in a micro‐RNA (hsa‐miR‐107) recognition motif, with a superior match for the risk (T) allele.In vitro expression studies revealed that CHGA mRNA expression is inversely dependent on miR‐107 abundance in cells and tissues. The effects of miR‐107 were seen with transfected chimeric luciferase/CHGA 3’‐UTR (C+87 versus +87T) reporters in cell lines and corroborated in studies of coupled in vitro transcription/translation of each 3’‐UTR/C+87T allele in CHGA cDNA.In vivo, we exploited mice with a “humanized”CHGA locus (T/T homozygotes) generated by BAC transgenesis. These mice treated with miR‐107 antagomir yielded prolonged, significant falls in SBP/DBP compared to the wild‐type mice.Conclusion: The 3’‐UTRmRNA:miRNA‐107 (cis:trans) interaction plays a role in the clinical manifestations of the CHGA 3’‐UTR associations with BP and nephropathy.Grant Funding Source: Supported by National Institutes of Health (SMV & DTO'C)

Hox complex analysis through BAC recombineering.

BAC transgenesis in mice has proved to be useful in exploring the regulatory mechanisms and functions of the Hox complexes. The large constructs used may include most of the relevant components of the cis-regulatory landscape. Manipulations can be accomplished without compromising the integrity of the endogenous complex which reduces the likelihood of producing confounding phenotypic abnormalities. The development of recombineering tools has been critical in providing the means necessary to make many types of precise and varied manipulations of these large constructs. Here, we will discuss the methodologies necessary to manipulate Hox complex BACs, generation of transgenic animals bearing these constructs and the utilization of these resources to address fundamental aspects of Hox biology.