Cryopreservation Protocols for Genetically Engineered Mice.

Protocols for cryopreservation of mouse embryos and sperm are important for preserving genetically engineered mice (GEMs) used in research to study human development and diseases. Embryo cryopreservation is mainly carried out using either of two protocols: controlled gradual cooling or vitrification. Sperm cryopreservation protocols include two methodologies that are commonly referred to as JAX and CARD. Quality-control measures are necessary to ensure that GEMs are properly cryopreserved so that they can be retrieved for future use. An archiving system is also important in keeping proper records of frozen sperm and embryos. Frozen embryos and sperm are now preferred over live mice for shipping to distant locations. This article describes detailed protocols used in cryopreservation of mouse embryos and sperm, as well as their retrieval to live mice. © 2021 U.S. Government. Sperm cryopreservation Basic Protocol 1: JAX protocol for sperm cryopreservation Support Protocol 1: JAX protocol for making sperm cryopreservation medium Basic Protocol 2: JAX protocol for IVF of mouse sperm Alternate Protocol 1: Modified CARD protocol for sperm cryopreservation Support Protocol 2: CARD protocol for making sperm cryopreservation medium Alternate Protocol 2: CARD protocol for IVF of mouse sperm Embryo cryopreservation Basic Protocol 3: Cryopreserving and thawing 2-cell embryos Alternate Protocol 3: Cryopreserving and thawing 8-cell to morula-stage embryos Surgical transfer of embryos Basic Protocol 4: Infundibulum transfer of 2-cell to morula-stage embryos.

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

Generation of gene knockout mice by ES cell microinjection.

This unit describes protocols used in the production of chimeric mice that are then used for the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols for the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipets for embryo and cell manipulations, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur.

TGF-beta receptor I conditional knockout mice develop spontaneous squamous cell carcinoma.

We generated a mouse model with a conditional deletion of TGF-beta signaling in the neurons by crossing TGF-beta receptor I (TbetaRI) floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 conditional knockout (COKO) mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. Transplantation of these tumors into athymic nude mice resulted in 62% tumorigenicity. To determine whether evasion of the immune response plays any role in this tumorigenesis, we analyzed the expression levels of receptors for interleukin-13 (mIL-13R), a key negative regulator of tumor immunosurveillance, and found that 33% of COKO tumors expressed the IL-13R alpha2 chain. Primary cultures of the SCCs expressing IL-13R alpha2 were sensitive to the cytotoxic effect of IL-13R-directed cytotoxin treatment. This is the first demonstration that loss of TbetaRI can lead to spontaneous tumor formation. These mice can serve as a unique mouse model of SCC to evaluate the tumorigenicity and effect of anti-cancer therapeutics.

Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III.

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.

Perinatal abrogation of Cdk5 expression in brain results in neuronal migration defects.

Cyclin-dependent kinase 5 (Cdk5) is essential for the proper development of the CNS, as is evident from the perinatal lethality of conventional Cdk5 knockout (Cdk5-/-) mice. Cdk5 is also implicated in numerous complex functions of the adult CNS such as synaptic transmission, synaptic plasticity, and neuronal signaling. To elucidate the molecular roles of Cdk5 in the adult CNS, we have abrogated neuronal expression of Cdk5 in perinatal mice by using a cre-loxP system. The Cdk5-loxP flanked mice were crossed with the cre-transgenic mice in which the cre expression is driven by the murine neurofilament-heavy chain promoter, resulting in generation of viable Cdk5 conditional knockout mice with the restricted deletion of the Cdk5 gene in specific neurons beginning around embryonic day 16.5. Twenty-five percent of the Cdk5 conditional knockout mice carrying the heterozygous cre allele had neuronal migration defects confined to brain areas where neuronal migration continues through the perinatal period. These results indicate that abrogation of Cdk5 expression in mature neurons results in a viable mouse model that offers further opportunities to investigate the molecular roles of Cdk5 in the adult CNS.

Endocrine expression of the active form of TGF-beta1 in the TGF-beta1 null mice fails to ameliorate lethal phenotype.

TGF-beta1 null mice die by 3 to 4 weeks of age due to a severe autoimmune-mediated multifocal inflammation resulting in multi-organ failure. To assess the therapeutic potential of circulating levels of active TGF-beta1, we generated mice with endocrine expression of active TGF-beta1 on a TGF-beta1 null background (TGF-beta1 (-/-/TG)) by crossing TGF-beta1(+/-) mice with transgenic mice (TG) that express recombinant TGF-beta1 specifically in the liver and secrete it in the blood. The TGF-beta1 (-/-/TG) mice exhibit a survival profile similar to the TGF-beta1 (-/-) mice indicating a failure to rescue the lethal phenotype. However, serum TGF-beta1 levels in the TGF-beta1 (-/-/TG) mice were restored to near normal levels with expression in all the tissues, notably in the kidney and spleen. Histopathology showed reduced inflammation in the target tissues, especially in the heart. Interestingly, unlike TGF-beta1 (-/-) mice, the TGF-beta1 (-/-/TG) mice have glomerulonephritis in their kidneys similar to the TG mice. Thus, the phenotype of TGF-beta1 (-/-/TG) animal model indicates the potential role of circulating active-TGF-beta1 in reducing inflammation, but its failure to rescue lethality in TGF-beta1 null mice indicates a critical autocrine role of TGF-beta1.