Production of Transgenic Mice by Pronuclear Microinjection.

Pronuclear microinjection remains the most widely used method for the germline modification of mice and other species. The method is conceptually quite simple and at least in rodents can produce transgenic offspring with relatively high efficiency. Here, we describe the various components of the production of transgenic mice including a detailed list of materials and equipment. We outline in detail the preparation of animals, the retrieval, culture and transfer of embryos, the preparation of DNA, and the microinjection process. We have added a substantial collection of notes with helpful suggestions that reflect the many years of experience we have using this technology and our continuing efforts to improve animal welfare and the efficiency of producing transgenics.

Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice.

The discovery of new gene editing tools in the past several years has moved the transgenic field to a new level. The traditional random transgenesis method by pronuclear microinjection has been largely replaced by targeted or site-specific transgenic technologies without the need of homologous recombination in embryonic stem (ES) cells. In this chapter, I describe detailed protocols of an integrase-based approach, trademarked as "TARGATT™" (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into a predetermined chromosomal locus with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Generating Genetically Engineered Mice Using a Spermatogonial Stem Cell-Mediated Method.

Mouse spermatogonial stem cells (SSCs) can be grown in culture for long periods. Cultured SSCs, also called germline stem (GS) cells, maintain themselves by self-renewing proliferation while retaining the ability to differentiate into sperm. Thus, when transplanted into the seminiferous tubules of a host mouse testis, they settle in the basal compartment of the tubules and establish spermatogenenic colonies. The sperm produced in the host are competent to produce offspring. This can be exploited for the generation of genetically modified mice, through the transplantation of genetically modified GS cells. In this section, we describe a method of genome editing-mediated GS cell modification and transplantation.

Constitutive expression of antimicrobial peptide PR-39 in transgenic mice significantly enhances resistance to bacterial infection and promotes growth.

Use of huge amounts of antibiotics in farm animal production has promoted the prevalence of antibiotic-resistant bacteria, which poses a serious threat to public health. Therefore, alternative approaches are needed to reduce or replace antibiotic usage in the food animal industry. PR-39 is a pig-derived proline-rich antimicrobial peptide that has a broad spectrum of antibacterial activity and a low propensity for development of resistance by microorganisms. To test whether ubiquitous expression of PR-39 in transgenic (TG) mice can increase resistance against bacterial infection, we generated TG mice that ubiquitously express a pig-derived antimicrobial peptide PR-39 and analyzed their growth and resistance to infection of the highly pathogenic Actinobacillus pleuropneumoniae (APP) isolated from swine. The growth performance was significantly increased in TG mice compared with their wild-type (WT) littermates. After the APP challenge, TG mice exhibited a significantly higher survival rate and significantly lower tissue bacterial load than WT littermates. Furthermore, the tissue lesion severity that resulted from APP infection was milder in TG mice than that in their WT littermates. This study provides a good foundation for the development of PR-39-expressing TG animals, which could reduce the use of antibiotics in the farm animal industry.

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1.

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5'- and 3'-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.

The increased expression of follicle-stimulating hormone leads to a decrease of fecundity in transgenic Large White female pigs.

Follicle-stimulating hormone (FSH) is a pituitary gonadotropin regulating reproduction in mammals. Overexpression of the exogenous FSHα/ß genes from Chinese Erhualian pigs improved female fecundity of transgenic (TG) mice and male spermatogenesis ability of Large White TG boars. Here, we investigated the impact of the exogenous FSHα/ß genes on female reproductive performance of Large White TG pigs. First, we identified the integration site of the exogenous FSHα/ß genes at 140,646,456 bp on chromosome 9 in these TG pigs using whole-genome sequencing. Then, we showed that TG gilts had higher levels of serum FSH and FSHß protein in pituitary while had a potentially lower number of born piglets than their wild-type half sibs. TG gilts grew healthily and normally without significant difference in growth and health parameters as compared to WT gilts. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly lower in TG gilts than in WT gilts at the age of 300 days. Taken together, we proposed that the overexpressed FSHα/ß transgenes could cause deteriorate fecundity via disturbing the normal expression of the endogenous reproduction-related genes in female pigs. Our findings provide insight into the effect of overexpression of FSHα/ß on female reproduction performance in pigs.

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.

BACKGROUND: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. RESULTS: Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring. CONCLUSIONS: Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods.

Genetic modification of almost any species is now possible using approaches based on targeted nucleases. These novel tools now bypass previous limited species windows, allowing precision nucleotide modification of the genome at high efficiency, rapidly and economically. Here we focus on the modification of the mouse genome; the mouse, with its short generation time and comparatively low maintenance/production costs is the perfect mammal with which to probe the genome to understand its functions and complexities. Further, using targeted nucleases combined with homologous recombination, it is now possible to precisely tailor the genome, creating models of human diseases and conditions directly and efficiently in zygotes derived from any mouse strain. Combined these approaches make it possible to sequentially and progressively refine mouse models to better reflect human disease, test and develop therapeutics. Here, we briefly review the strategies involved in designing targeted nucleases (sgRNAs) providing solutions and outlining in detail the practical processes involved in precision targeting and modification of the mouse genome and the establishing of new precision genetically modified mouse lines.

Retrotransposition creates sloping shores: a graded influence of hypomethylated CpG islands on flanking CpG sites.

Long interspersed elements (LINEs), through both self-mobilization and trans-mobilization of short interspersed elements and processed pseudogenes, have made an indelible impact on the structure and function of the human genome. One consequence is the creation of new CpG islands (CGIs). In fact, more than half of all CGIs in the genome are associated with repetitive DNA, three-quarters of which are derived from retrotransposons. However, little is known about the epigenetic impact of newly inserted CGIs. We utilized a transgenic LINE-1 mouse model and tracked DNA methylation dynamics of individual germline insertions during mouse development. The retrotransposed GFP marker sequence, a strong CGI, is hypomethylated in male germ cells but hypermethylated in somatic tissues, regardless of genomic location. The GFP marker is similarly methylated when delivered into the genome via the Sleeping Beauty DNA transposon, suggesting that the observed methylation pattern may be independent of the mode of insertion. Comparative analyses between insertion- and non-insertion-containing alleles further reveal a graded influence of the retrotransposed CGI on flanking CpG sites, a phenomenon that we described as "sloping shores." Computational analyses of human and mouse methylomic data at single-base resolution confirm that sloping shores are universal for hypomethylated CGIs in sperm and somatic tissues. Additionally, the slope of a hypomethylated CGI can be affected by closely positioned CGI neighbors. Finally, by tracing sloping shore dynamics through embryonic and germ cell reprogramming, we found evidence of bookmarking, a mechanism that likely determines which CGIs will be eventually hyper- or hypomethylated.

Growth retardation and hair loss in transgenic mice overexpressing human H-ferritin gene.

H-ferritin (HF) is a core subunit of the iron storage protein ferritin, and plays a central role in the regulation of cellular iron homeostasis. Recent studies revealed that ferritin and HF are involved in a wide variety of iron-independent functions, including regulating biological processes during physiological and pathological conditions, and can be overexpressed in some human diseases. To investigate the in vivo function of HF, we generated transgenic (tg) mice overexpressing the human HF gene (hHF-tg). We established two independent hHF-tg mouse lines. Although both lines of hHF-tg mice were viable, they showed reduced body size compared to wild-type (WT) mice at 4-12 weeks of age. Serum iron concentration and blood parameters of hHF-tg mice such as hemoglobin and red blood cell counts were comparable to those of WT mice. At 3-5 weeks of age, hHF-tg mice exhibited temporary loss of coat hair on the trunk, but not on the head or face. Histological analyses revealed that although initial hair development was normal, hHF-tg mice had epidermal hyperplasia with hyperkeratosis, dilated hair follicles, bended hair shafts and keratinous debris during the hairless period. In conclusion, we showed that hHF-tg mice exhibited mild growth retardation and temporary hairless phenotype. Our findings highlight the physiological roles of HF and demonstrate that hHF-tg mice are useful for understanding the in vivo functions of HF.

Androgenetic haploid embryonic stem cells produce live transgenic mice.

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

Cardioprotection by Hepc1 in cTnT(R141W) transgenic mice.

Hepcidin 1 (Hepc1) is a peptide hormone secreted by the liver in response to iron loading. It is expressed in the heart and is thought to play a role in the regulation of iron homeostasis in an autocrine and paracrine fashion. We have shown that expression of Hepc1 is strongly down-regulated in the heart of the cTnT(R141W) transgenic mouse model of dilated cardiomyopathy (DCM) at 3 months of age. Transgenic mice with heart tissue-specific Hepc1 expression alone or in combination with the cTnT(R141W) mutation were produced to study the effects of Hepc1 on DCM. Transgenic expression of Hepc1 was found to be nonlethal and resulted in decreased mortality in cTnT(R141W) transgenic mice, from 29.6 to 7.4%(n  = 27; P < 0.05), through 7 months of age. Expression of Hepc1 also brought about increases in the left ventricular wall, as well as ejection fraction and fractional shortening. In addition, the expression of Hepc1 inhibited the fibrosis and ultra-structural alterations seen in cTnT(R141W) transgenic mice. Furthermore, transgenic expression of Hepc1 restored the iron level and phosphorylation level of extracellular signal-regulated kinases 1/2 (ERK1/2) in the heart tissues of cTnT(R141W) transgenic mice. It was concluded that transgenic expression of Hepc1 compensated for the loss of Hepc1 expression and the release of iron and brought about a marked improvement in the pathologic phenotype of DCM, in which the ERK1/2 signal pathway might play an important role.

Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b hnn stems from abnormal Shh signaling.

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b(hnn), to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b(hnn) mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b(hnn) dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b(hnn) mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b(hnn) embryos indirectly causes dorsal defects.

Pancreatic ß-cells are generated by neogenesis from non-ß-cells after birth.

The mass of pancreatic ß-cells is maintained throughout lifetime to control blood glucose levels. Although the major mechanism of the maintenance of ß-cell mass after birth is thought to be selfreplication of pre-existing ß-cells, it is possible that pancreatic ß-cells are also generated from non-ß-cells. Here, we address this issue by using the inducible Cre/loxP system to trace ß-cells. We generated Ins2-CreERT2/R26R-YFP double knock-in mice, in which pancreatic ß-cells can be labeled specifically and permanently upon injection of the synthetic estrogen analog tamoxifien, and then traced the ß-cells by pulse and chase experiment in several different conditions. When ß-cells were labeled in adults under physiological and untreated conditions, the frequency of the labeling (labeling index) was not altered significantly throughout the 12-month experimental period. In addition, the labeling index was not changed after ablation of ß-cells by streptozotocin treatment. However, when tamoxifen was injected to pregnant mothers just before they gave birth, the labeling index in the neonates was decreased significantly around weaning, suggesting that ß-cells are generated from non-ß-cells. These results indicate that various mechanisms are involved in the maintenance of ß-cells after birth, and that the present system using knock-in mice is useful for investigation of ß-cell fate.

Insights from mouse models to understand neurodegeneration in Down syndrome.

Individuals with trisomy 21, also known as Down syndrome (DS), develop a clinical syndrome including almost identical neuropathological characteristics of Alzheimer's disease (AD) observed in non-DS individuals. The main difference is the early age of onset of AD pathology in individuals with DS, with hish incidence of clinical symptoms in the late 40- early 50 years of age. The neuropathology of AD in persons with DS is superimposed with the developmental abnormalities causing alterations of neuronal morphology and function. Despite the ubiquitous occurrence of AD neuropathology, clinical signs of dementia do not occur in all adults with DS even at older ages. Phenotype analysis of DS mouse models has revealed a differential age-related neurodegenerative pattern that correlates with specific biochemical and molecular alterations at the cellular level. In fact, several individual genes found in trisomy in DS have been functionally related to neuronal degeneration. Thus, mouse models overexpressing HSA21 gene(s) are fundamental to understand the neurodegenerative process in DS, as described in the present review. In addition, these models might allow to define and evaluate potential drug targets and to develop therapeutic strategies that may interfere or delay the onset of AD.