Bacterial Artificial Chromosome Recombineering Research Articles

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research.

Herpesviruses are major pathogens that infect humans and animals. Manipulating the large genome is critical for exploring the function of specific genes and studying the pathogenesis of herpesviruses and developing novel anti-viral vaccines and therapeutics. Bacterial artificial chromosome (BAC) technology significantly advanced the capacity of herpesviruses researchers to manipulate the virus genomes. In the past years, advancements in BAC-based genome manipulating and screening strategies of recombinant BACs have been achieved, which has promoted the study of the herpes virus. This review summarizes the advances in BAC-based gene editing technology and selection strategies. The merits and drawbacks of BAC-based herpesvirus genome editing methods and the application of BAC-based genome manipulation in viral research are also discussed. This review provides references relevant for researchers in selecting gene editing methods in herpes virus research. Despite the achievements in the genome manipulation of the herpes viruses, the efficiency of BAC-based genome manipulation is still not satisfactory. This review also highlights the need for developing more efficient genome-manipulating methods for herpes viruses.

Development of a Next-Generation Vaccine Platform for Porcine Epidemic Diarrhea Virus Using a Reverse Genetics System.

For the past three decades, the porcine epidemic diarrhea virus (PEDV) has remained an enormous threat to the South Korean swine industry. The scarcity of an effective method for manipulating viral genomes has impeded research progress in PEDV biology and vaccinology. Here, we report the development of reverse genetics systems using two novel infectious full-length cDNA clones of a Korean highly pathogenic-G2b strain, KNU-141112, and its live attenuated vaccine strain, S DEL5/ORF3, in a bacterial artificial chromosome (BAC) under the control of a eukaryotic promoter. Direct transfection of cells with each recombinant BAC clone induced cytopathic effects and produced infectious progeny. The reconstituted viruses, icKNU-141112 and icS DEL5/ORF3, harboring genetic markers, displayed phenotypic and genotypic properties identical to their respective parental viruses. Using the DNA-launched KNU-141112 infectious cDNA clone as a backbone, two types of recombinant viruses were generated. First, we edited the open reading frame 3 (ORF3) gene, as cell-adapted strains lose full-length ORF3, and replaced this region with an enhanced green fluorescent protein (EGFP) gene to generate icPEDV-EGFP. This mutant virus presented parental virus-like growth kinetics and stably retained robust EGFP expression, indicating that ORF3 is dispensable for PEDV replication in cell culture and is a tolerant location for exogeneous gene acceptance. However, the plaque size and syncytia phenotypes of ORF3-null icPEDV-EGFP were larger than those of icKNU-141112 but similar to ORF3-null icS DEL5/ORF3, suggesting a potential role of ORF3 in PEDV cytopathology. Second, we substituted the spike (S) gene with a heterologous S protein, designated S51, from a variant of interest (VOI), which was the most genetically and phylogenetically distant from KNU-141112. The infectious recombinant VOI, named icPEDV-S51, could be recovered, and the rescued virus showed indistinguishable growth characteristics compared to icKNU-141112. Virus cross-neutralization and structural analyses revealed antigenic differences in S between icKNU-141112 and icPEDV-S51, suggesting that genetic and conformational changes mapped within the neutralizing epitopes of S51 could impair the neutralization capacity and cause considerable immune evasion. Collectively, while the established molecular clones afford convenient, versatile platforms for PEDV genome manipulation, allowing for corroborating the molecular basis of viral replication and pathogenesis, they also provide key infrastructural frameworks for developing new vaccines and coronaviral vectors.

Plant Gene Modification by BAC Recombineering.

Recombineering approaches exploiting the bacteriophage λ Red recombination functions are widely used for versatile modification of eukaryotic genes carried by bacterial artificial chromosomes (BACs) in E. coli. Whereas BAC transformation provides a simple means for integration of modified genes into the genomes of animal cells to generate knock-in and knockout lines, successful application of this strategy is hampered by low frequency of homologous recombination in higher plants. However, plant cells can be transformed at a high frequency using the transferred DNA (T-DNA) of Agrobacterium, which is stably and randomly integrated into the plant genome. The function of plant genes that are modified by recombineering and transferred by Agrobacterium T-DNA vectors into plant cells can thus be suitably studied using genetic complementation of knockout mutations induced by either T-DNA insertions or genome editing with T-DNA-based Crisp/Cas9 constructs. Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position. The second "turbo"-recombineering protocol exploits various I-SceI insertion cassettes for fusing of fluorescent protein tags to the plant gene products to facilitate the characterization of their in vivo interacting partners by affinity purification, mass spectrometry, and cellular localization studies.

BAC Recombineering and Transgenesis to Study Cell Polarity and Polarized Tissue Morphogenesis in Mice.

Planar cell polarity (PCP) signaling plays a critical role in coordinating cell polarity during various organogenesis processes in mammals, and its disruption is causal to numerous congenital disorders in humans. To elucidate its actions in mammals, mouse genetics is an indispensable approach. Given that both gain- and loss-of-function of many PCP genes often cause similar defects, the standard mouse transgenic approach may not always be ideal for studying PCP genes in their wild-type and mutant forms. Here we describe using BAC (bacterial artificial chromosomes) transgenes as a versatile and effective alternative. Transgenes made from BACs, which are genomic clones 100-200kb in size, can more faithful recapitulate endogenous gene expression levels and patterns. Bacterial based recombination system can be used to efficiently introduce mutations, fluorescent protein tags, and LoxP sites for conditional expressions. Cre can also be inserted into BACs to map the contribution of cells expressing any PCP gene of interest, and study PCP mediated tissue morphogenesis.

Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone. Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days. Overall, our new strategy may provide a new avenue for BAC recombineering, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

Establishment of a Simple and Efficient Reverse Genetics System for Canine Adenoviruses Using Bacterial Artificial Chromosomes.

Canine adenoviruses (CAdVs) are divided into pathotypes CAdV1 and CAdV2, which cause infectious hepatitis and laryngotracheitis in canid animals, respectively. They can be the backbones of viral vectors that could be applied in recombinant vaccines or for gene transfer in dogs and in serologically naïve humans. Although conventional plasmid-based reverse genetics systems can be used to construct CAdV vectors, their large genome size creates technical difficulties in gene cloning and manipulation. In this study, we established an improved reverse genetics system for CAdVs using bacterial artificial chromosomes (BACs), in which genetic modifications can be efficiently and simply made through BAC recombineering. To validate the utility of this system, we used it to generate CAdV2 with the early region 1 gene deleted. This mutant was robustly generated and attenuated in cell culture. The results suggest that our established BAC-based reverse genetics system for CAdVs would be a useful and powerful tool for basic and advanced practical studies with these viruses.

OHSV Genome Editing by Means of galK Recombineering.

Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.

Characterization of a BAC transgenic mouse expressing Krt19-driven iCre recombinase in its digestive organs.

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, β-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.

Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches

Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3′ of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment.

Abstract 5097: Knock-in of a human tumor suppressor (25-kilobase pairs) by traditional and CRISPR/Cas9-stimulated homologous recombination

Abstract Here we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model inserting a 25-kbp segment of a human tumor-suppressor gene in place of 17 kbp of its corresponding mouse orthologue. Included within the human fragment is a conditionally removable (by Cre-loxP) segment of the gene's second intron, a cryptic exon immediately 3' of this, and a native exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides and Cas9 into mouse zygotes (CRISPR approach). In both instances, we were able to achieve humanization of the tumor suppressor to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, intronic segment. Citation Format: Tiffany Leidy-Davis, Kai Cheng, Leslie Goodwin, Judith Morgan, Wen Chun Juan, Xavier Roca, Sin-Tiong Ong, David E. Bergstrom. Knock-in of a human tumor suppressor (25-kilobase pairs) by traditional and CRISPR/Cas9-stimulated homologous recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5097.

Identification of novel cis-regulatory elements of Eya1 in Xenopus laevis using BAC recombineering

The multifunctional Eya1 protein plays important roles during the development of cranial sensory organs and ganglia, kidneys, hypaxial muscles and several other organs in vertebrates. Eya1 is encoded by a complex locus with candidate cis-regulatory elements distributed over a 329 kbp wide genomic region in Xenopus. Consequently, very little is currently known about how expression of Eya1 is controlled by upstream regulators. Here we use a library of Xenopus tropicalis genomic sequences in bacterial artificial chromosomes (BAC) to analyze the genomic region surrounding the Eya1 locus for enhancer activity. We used BAC recombineering to first create GFP reporter constructs, which were analysed for enhancer activity by injection into Xenopus laevis embryos. We then used a second round of BAC recombineering to create deletion constructs of these BAC reporters to localize enhancer activity more precisely. This double recombineering approach allowed us to probe a large genomic region for enhancer activity without assumptions on sequence conservation. Using this approach we were able to identify two novel cis-regulatory regions, which direct Eya1 expression to the somites, pharyngeal pouches, the preplacodal ectoderm (the common precursor region of many cranial sensory organs and ganglia), and other ectodermal domains.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts.

Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes.

Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle-driven Enrichment (FPE), a method based on the population genetics "founder principle," to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc.

Heterologous expression of Avermectins biosynthetic gene cluster by construction of a Bacterial Artificial Chromosome library of the producers

Avermectins, a group of polyketide natural products, are widely used as anthelmintics in agriculture. Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives, including Ivermectin and Doramectin. It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo. Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro. We constructed a Bacterial Artificial Chromosome (BAC) library of S. avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb. Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave (81 Kb in size) were screened out from the library. Then, ave was hetero-expressed in S. lividans. Three Avermectin components, A2a, B1a and A1a were detected from the cell extracts of recombinant strains. It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.

Protective efficacy of a recombinant bacterial artificial chromosome clone of a very virulent Marek’s disease virus containing a reticuloendotheliosis virus long terminal repeat

ABSTRACTMarek’s disease virus (MDV), an alphaherpesvirus, causes Marek’s disease (MD), a lymphoproliferative disease in poultry characterized by T-cell lymphomas, nerve lesions, and mortality. Vaccination is used worldwide to control MD, but increasingly virulent field strains can overcome this protection, driving a need to create new vaccines. Previous studies revealed that insertion of reticuloendotheliosis virus (REV) long terminal repeat (LTR) into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, rendered the resultant recombinant virus, rMd5 REV-LTR BAC, fully attenuated in maternal antibody positive (Mab+) chickens at passage 40. In the current study, the protective efficacy of rMd5 REV-LTR BAC was evaluated. First, passage 70 was identified as being fully attenuated in maternal antibody negative chickens and chosen as the optimal passage level for use in protective efficacy studies. Second, three protective efficacy trials were conducted comparing the rMd5 REV-LTR p70 BAC to the CVI988/Rispens vaccine. Groups of Mab+ and Mab− 15I5 × 71 chickens were vaccinated in ovo at 18 days of embryonation or intra-abdominally at day of hatch, and challenged at 5 days post-hatch with the vv+MDV strain 686. Vaccination at day of hatch and in ovo with rMd5 REV-LTR p70 BAC protected chickens against MDV-induced bursa and thymic atrophy, but did not provide the same level of protection against MD tumours as that afforded by the commercial vaccine, CVI988/Rispens.

Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located −1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17β-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The −1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the −1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding.

Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR).

A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described. We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette. The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step.