Generation of a Twist1 conditional null allele in the mouse

Twist1 is the mouse ortholog of TWIST1, the human gene mutated in Saethre-Chotzen syndrome. Previously, a Twist1 null allele was generated by gene targeting in mouse embryonic stem cells. Twist1 heterozygous mice develop polydactyly and a craniofacial phenotype similar to Saethre-Chotzen patients. Mice homozygous for the Twist1 null allele die around embryonic day 11.5 (E11.5) with cranial neural tube closure and vascular defects, hindering in vivo studies of Twist1 function at later stages of development. Here, we report the generation of a Twist1 conditional null allele in mice that functions like a wild-type allele but can be converted to a null allele upon Cre-mediated recombination.     

Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice.

The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human alpha-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus.     

Generation of new Notch2 mutant alleles

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. We previously described construction and analysis of a hypomorphic allele of the Notch2 gene. Homozygosity for this allele leads to embryonic and perinatal lethality due to cardiovascular and kidney defects. We report here novel Notch2 mutant alleles generated by gene targeting in embryonic stem cells, including a conditional null allele in which exon 3 of the Notch2 gene is flanked by loxP sequences. These new Notch2 mutant alleles expand the set of tools available for studying the myriad roles of the Notch pathway during mammalian development and will enable analysis of Notch2 function at additional stages of embryogenesis and in adult mice.     

Gene trap and gene inversion methods for conditional gene inactivation in the mouse

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.     

Generation of a conditional disruption of the Tsc2 gene

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 gene. Patients afflicted with TSC develop tumors in various organ systems, but cerebral pathology is particularly severe. Conventional gene disruption of the Tsc1 or Tsc2 gene in mice cause limited central nervous system pathology. Homozygous deletion of either gene causes midgestation lethality. To circumvent the homozygous lethality of the conventional Tsc2 knockout we have generated a conditional allele of the Tsc2 gene by homologous recombination in mouse ES cells. The homozygous Tsc2(flox/flox) mice are identical to wildtype in many organs typically affected by TSC, especially the brain. Using this Tsc2(flox) allele we have generated a null allele using Cre recombination. This allele will be useful in investigating TSC pathology with appropriate cell and organ specific Cre-transgenic mice.     

Generation of a Bmp2 conditional null allele

Bone morphogenetic proteins (Bmp's) are known to play many important roles in embryogenesis. In addition, recent data from human genetic studies has revealed that Bmp's also have important functions in maintenance of the adult phenotype and aging. The original Bmp2 germline null allele resulted in lethality at embryonic day 7.0-10.5 due to malformation of the amnion/chorion and cardiac malformations. Because the early embryonic lethality of the Bmp2 germline null allele hinders further investigation into Bmp2 function at later stages, we generated a Bmp2 conditional null allele. Using gene targeting in mouse embryonic stem (ES) cells, we introduced LoxP sites upstream and downstream of Bmp2 exon 3 that encodes the mature peptide. Our results indicate that the Bmp2 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after cre recombinase-induced recombination.     

Cloning,ligand-binding,and temporal expression of ecdysteroid receptors in the diamondback moth,Plutella xylostella

Background

RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.

Results

Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.

Conclusions

Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.     

A simplified strategy for titrating gene expression reveals new relationships between genotype,environment, and bacterial growth

A lack of high-throughput techniques for making titrated, gene-specific changes in expression limits our understanding of the relationship between gene expression and cell phenotype. Here, we present a generalizable approach for quantifying growth rate as a function of titrated changes in gene expression level. The approach works by performing CRISPRi with a series of mutated single guide RNAs (sgRNAs) that modulate gene expression. To evaluate sgRNA mutation strategies, we constructed a library of 5927 sgRNAs targeting 88 genes in Escherichia coli MG1655 and measured the effects on growth rate. We found that a compounding mutational strategy, through which mutations are incrementally added to the sgRNA, presented a straightforward way to generate a monotonic and gradated relationship between mutation number and growth rate effect. We also implemented molecular barcoding to detect and correct for mutations that ‘escape’ the CRISPRi targeting machinery; this strategy unmasked deleterious growth rate effects obscured by the standard approach of ignoring escapers. Finally, we performed controlled environmental variations and observed that many gene-by-environment interactions go completely undetected at the limit of maximum knockdown, but instead manifest at intermediate expression perturbation strengths. Overall, our work provides an experimental platform for quantifying the phenotypic response to gene expression variation.     

Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications

Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating thein vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.Abbreviations ES embryonic stem - neo^r neomycin resistance gene - HSV herpes simplex virus - tk thymidine kinase gene - PCR polymerase chain reaction - LIF leukemia inhibitory factor - LTP long-term potentiation - Rb retinoblastoma gene product - CF cystic fibrosis     

Generation of a conditional null allele of jumonji

The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the alpha-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.     

Generation of an Frs2alpha conditional null allele

The fibroblast growth factor (FGF) signaling family controls a broad spectrum of cellular processes in development and adult tissue homeostasis and function, which is expressed in almost all tissues at all stages. FGF receptor substrate 2 alpha (FRS2alpha) is an adaptor protein that recruits downstream substrates to the FGF receptor (FGFR) tyrosine kinase. Disruption of Frs2alpha gene in mice abrogates activation of the mitogen-activated protein kinase pathway by the FGFR and leads to embryonic lethality at day E7.5 post copulation. To circumvent the embryonic lethality resulting from disruption of the Frs2alpha gene, which hinders further characterization of the role of FRS2alpha in adult tissue function and homeostasis, we generated an Frs2alpha conditional null allele for temporally- and tissue-specific disruption of the Frs2alpha gene. Using gene targeting in mouse embryonic stem cells, we introduced two loxP sites flanking the largest coding exon, exon 5, in the Frs2alpha allele. Our results indicate that the floxed Frs2alpha (Frs2alpha(flox)) allele is a true conditional null allele that encodes wildtype activity and is converted to a null allele after Cre recombinase mediated recombination.     

Genetic knockouts and knockins in human somatic cells

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.     

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.     

Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability

The rapamycin-insensitive mTOR complex 2 (mTORC2) has been suggested to play an important role in growth factor-dependent signaling. To explore this possibility further in a mammalian model system, we disrupted the expression of rictor, a specific component of mTORC2, in mice by using a multiallelic gene targeting strategy. Embryos that lack rictor develop normally until E9.5, and then exhibit growth arrest and die by E11.5. Although placental defects occur in null embryos, an epiblast-specific knockout of rictor only delayed lethality by a few days, thereby suggesting other important roles for this complex in the embryo proper. Analyses of rictor null embryos and fibroblasts indicate that mTORC2 is a primary kinase for Ser473 of Akt/PKB. Rictor null fibroblasts exhibit low proliferation rates, impaired Akt/PKB activity, and diminished metabolic activity. Taken together, these findings indicate that both rictor and mTORC2 are essential for the development of both embryonic and extraembryonic tissues.     

Requirement of CDC45 for Postimplantation Mouse Development

CDC45 is required for the initiation of DNA replication in Saccharomyces cerevisiae and functions as a DNA polymerase alpha loading factor in Xenopus, but its role in mammalian DNA replication is unknown. To investigate the genetic and physiological functions of CDC45, we used a gene targeting strategy to generate mice lacking a functional CDC45 gene. Homozygous mutant mice lacking a functional CDC45 gene underwent uterine implantation and induced uterine decidualization but did not develop substantially thereafter. Detailed analysis of CDC45 null embryos cultured in vitro revealed impaired proliferation of the inner cell mass. These findings make CDC45 the only putative replication factor experimentally proven to be essential for mammalian development. The CDC45 gene localizes to human chromosome 22q11.2 in the DiGeorge syndrome critical region (DGCR). Almost 90% of individuals with congenital cardiac and craniofacial defects have a monoallelic deletion in the DGCR that includes CDC45. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities, so that it is unlikely that hemizygosity of CDC45 alone is responsible for the cardiac and craniofacial defects in the congenital syndromes.