Insertional Mutation on Mouse Chromosome 18 with Vestibular and Craniofacial Abnormalities

A dominant mutation was generated in transgenic mice as a consequence of insertional mutation. Heterozygous mice from transgenic line 9257 (Tg(9257)) are hyperactive with bidirectional circling behavior and have a distinctive facial appearance due to hypoplasia of the nasal bone. Morphological analysis of the inner ear revealed asymmetric abnormalities of the horizontal canal and flattening or invagination of the crista ampullaris, which can account for the circling behavior. The sensory epithelium appeared to be normal. The transgene insertion site was localized by in situ hybridization to the B1 band of mouse chromosome 18. Genetic mapping in an interspecific backcross demonstrated the gene order centromere--Tg(9257)--8.8 +/- 3.4--Grl-1, Egr-1, Fgf-1, Apc--14.7 +/- 4.3--Pdgfr. The phenotype and the mapping data suggest that the transgene may be inserted at the Twirler locus. Homozygosity for the transgene results in prenatal lethality, but compound heterozygotes carrying the Tw allele and the transgene are viable. The function of the closely linked ataxia locus is not disrupted by the transgene insertion. This insertional mutant will provide molecular access to genes located in the Twirler region of mouse chromosome 18.     

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.     

H beta 58, an insertional mutation affecting early postimplantation development of the mouse embryo

The generation and analysis of insertional mutations affecting mouse embryogenesis provides a powerful method to identify new genes that function in early development. In this paper, we describe an insertional mutation that interferes with postimplantation mouse development beginning at the time of gastrulation. Embryos homozygous for the H beta 58 transgenic insertion developed normally through the early postimplantation, egg cylinder stage (day 6.5 of development). At the primitive streak stage (day 7.5), however, they began to display characteristic abnormalities, including a retardation in the growth of the embryonic ectoderm (the earliest identifiable defect), and in some cases abnormalities of the amnion and chorion. Homozygotes continued to develop for 2-3 more days, reaching the size of a normal 8.5 day embryo, and formed tissues representative of all three germ layers, including several differentiated cell types. The site of insertion was mapped, by a combination of cytogenetic and genetic methods, to chromosome 10, and it appeared to define a new genetic locus. The inserted transgene provided a probe to clone and characterize the mutant locus, as well as the corresponding wild-type locus. In addition to an insertion of 10-20 copies of the transgene, the mutant locus contained a deletion of 2-3 kb of DNA found at the wild-type locus, and possibly an insertion of mouse repetitive DNA. However, genomic sequences on both sides of the insertion site remained co-linear in the wild-type and mutant genomes, and no chromosomal abnormalities could be detected. Five single copy DNA probes spanning the insertion site were tested for their ability to hybridize to RNA from 8.5 day embryos; one of the probes (located within the region deleted from the mutant chromosome) hybridized to a 2.7 kb mRNA encoded at the H beta 58 locus, thus identifying a gene whose disruption appears to be responsible for the mutant phenotype.     

Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice.

In line 4 transgenic mice, six to eight copies of a 50-kb lambda recombinant clone are arranged in a head-to-tail tandem array on chromosome 3. Embryos homozygous for the transgene become arrested in their development on Day 5 of gestation shortly after implantation. The insertion site was cloned using a small segment of the transgene as a probe. Comparison of the insertion site with the wildtype locus indicates that a 22-kb deletion of host DNA has occurred in line 4 mice. Restriction enzyme analyses showed that neither the tandem array nor the flanking chromosomal DNA had any detectable rearrangements. Sequencing of the junctions between host and foreign DNA, however, revealed insertions of small fragments of DNA of unknown origin as well as an insertion of a DNA segment derived from another region of the transgene. Therefore, disruption of an essential gene in the line 4 transgenic mouse may have been caused by the insertion of 300-400 kb of foreign DNA or a deletion of sequences in the host genome.     

A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.     

Identifying and genotyping transgene integration loci

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.     

A 15-base pair (bp) palindromic insertion associated with a 3-bp deletion in exon 10 of the gp91-phox gene,detected in two patients with X-linked chronic granulomatous disease

Molecular genetic studies were carried out on two maternal cousins with X-linked chronic granulomatous disease (X-CGD). Sequencing analysis of polymerase chain reaction (PCR)-amplified DNA fragments from both patients revealed a 15-base pair (bp) insertion associated with a 3-bp deletion in exon 10 of the cytochrome b heavy chain (gp91-phox) gene. Results of genomic PCR with primers flanking the insertion/deletion site confirmed the mutation, and also demonstrated that their mothers were carriers for the disease. Palindromic sequences were found in the 15-bp insertion as well as in the flanking 3-bp deletion site, which may play a role in the mechanism of this mutation.     

Chromosomal localization of the retinol binding protein gene and its elimination as a candidate gene for the repeated epilation (Er) mutation in mice.

The repeated epilation (Er) mutation is an autosomal defect that blocks differentiation in stratified epithelia and appendages in mice. Plasma retinol binding protein (RBP) was tested as a possible candidate gene for the Er defect because of the importance of retinol as a modulator of epithelial morphogenesis and differentiation. Two approaches were used: (1) cloning and sequencing of the RBP cDNA from normal and mutant mice, and (2) the chromosomal localization of the mouse RBP gene. The mouse RBP sequence differs slightly from that of the rat RBP, but mutant and normal mouse RBP have identical sequences. The mouse RBP gene was localized by in situ hybridization to the distal portion of chromosome 19. This physical mapping confirms the recent assignment of the gene to chromosome 19 by linkage analysis. These results eliminate the RBP gene as a candidate gene for the defect in the Er mutation that maps to chromosome 4.     

Transgenic Mouse Model of Hemifacial Microsomia: Cloning and Characterization of Insertional Mutation Region on Chromosome 10

The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5′ and 3′ flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggest that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans.     

水稻剑叶突变体的表型及T-DNA旁邻基因分析

从已构建的水稻（Oryza sativa L.）T-DNA插入突变体中鉴定获得一株穗部额外发育出叶片的突变体,并根据该叶片的形态学位置将其命名为剑叶突变体（J4）。研究表明这种额外发育的叶片呈现明显的缺陷,主要表现为叶片短小、表皮细胞变小、叶片中维管束数目减少等。进一步通过TAIL-PCR和inverse-PCR的方法克隆该突变体中T-DNA插入位置的旁邻序列,从而准确地将T-DNA定位到2号染色体上。基因表达分析显示,T-DNA插入位置附近的AK100376基因在J4突变体以及表型类似突变体neck leaf 1中的表达均被明显下调,可初步将其确定为与剑叶突变体表型相关的候选基因。     

Ectopic expression of chloramphenicol acetyltransferase (CAT) in the cerebellum in mice transgenic for a carbonic anhydrase II promoter-CAT construct that is without apparent phenotypic effect

We have developed six transgenic lines of mice with constructs containing presumptive 5' regulatory regions of carbonic anhydrase II (CA II). Four of the lines contained 1,100 bases of the 5' flanking region of the human CA II gene, and two transgenic lines resulted from a construct containing 500 bases of the 5' flanking region of the mouse CA II gene. Tissue-specific expression of the chloramphenicol acetyltransferase (CAT) gene was not obtained in any of the transgenic lines. One of the transgenic lines was found to have high levels of expression of CAT in cerebellum. This expression persisted through multiple generations and was independent of the parental origin of the transgene. On the assumption that the expression was due to the insertion of the transgene in or near a gene expressed normally in cerebellum, homozygous mice were bred for the transgenic insert to see if a mutation might have been induced. Homozygous mice were found and seemed to be normal in all aspects of their phenotype studied. Thus, in this case, neither the insertion of the gene nor the ectopic expression of CAT seemed to be harmful to the animals.     

Identification of a gene, included in control of root system development in Arabidopsis thaliana

Genetic and molecular genetic analysis of a lethal root mutant of Arabidopsis thaliana was carried out. The mutant was obtained from a collection created earlier by means of insertion mutagenesis. The mutation was found to be recessive. It was caused by an insertion of the T region of vector pLD3 used for transformation of germinating seeds when creating the collection of insertion mutants. A 118-bp DNA fragment flanking the left border of the insertion was isolated using the TAIL PCR technique, and its nucleotide sequence was determined. Computer analysis of this DNA region demonstrated that it was located in exon 32 of the YUP8HI2R.44 gene in chromosome 1.     

水稻基因组上一个Ds切离及其双位点插入行为的分子鉴定

玉米转座元件Ac/Ds是hAT转座子家族的成员, 导入水稻基因组后具有转座活性, 尽管转座机制还不完全清楚, 但它们通常经保守的非复制型“剪切-粘贴”过程转座。研究表明, 在Ac编码的转座酶作用下, Ds从原位点切离后常优先重新插入到连锁位点。文章利用TAIL-PCR技术从水稻一个Ds插入突变体及其回复突变体中分离Ds侧翼序列, 结合生物信息学分析方法, 对Ds在突变体上插入位点、回复突变体内切离足迹和重新插入位点进行了分子鉴定。结果显示, 突变体中Ds从3号染色体切离后, 在原插入位点残留了8 bp足迹序列(CATCATGA), 引起Ds标记基因外显子和内含子数目增加, 从而影响基因结构。切离后的Ds重新插入回复突变体第2和第6号染色体上, 分别编码烟草胺氨基转移酶和衰老相关蛋白的2个基因的编码区。因此, 典型的“剪切-粘贴”机制不能完全解释Ds的转座行为, Ds转座存在“剪切-复制-粘贴”的特点。     

Mouse silver mutation is caused by a single base insertion in the putative cytoplasmic domain of Pmel 17.

This laboratory has established in previous studies that Pmel 17, a gene expressed specifically in melanocytes, maps near the silver coat color locus (si/si) on mouse chromosome 10. In the current study, we have focused on determining whether or not the si allele carries a mutation in Pmel 17. Pmel 17 cDNA clones, isolated from wild-type and si/si murine melanocyte cDNA libraries, were sequenced and compared. A single nucleotide (A) insertion was found in the putative cytoplasmic tail of the si/si Pmel 17 cDNA clone. This insertion is predicted to alter the last 24 amino acids at the C-terminus. Also predicted is the extension of the Pmel 17 protein by 12 residues because a new termination signal created downstream from the wild-type reading frame. The mutation was confirmed by the sequence of the PCR-amplified genomic region flanking and including the mutation site. The fact that si/si Pmel 17 was not recognized by antibodies directed toward the C-terminal 15 amino acids of wild-type Pmel 17, indicated a defect in this region. We conclude from these results that silver pmel 17 protein has a major defect at the carboxyl terminus. The chromosomal location and the identification of a potentially pathologic mutation in si-Pmel 17 support our conclusion that Pmel 17 is encoded at the silver locus.     

Allocation of a transgenic c-myc integration site to mouse chromosome 8B3-C1

A recombinant plasmid containing the mouse c-myc gene was injected into mouse pronuclei. The transgenic line 478 contains about 100 copies of the transgene integrated into one chromosome site. By in situ hybridization, the integration site was localized to chromosome 8B3-C1.     

Somatic gene targeting with RNA/DNA chimeric oligonucleotides: an analysis with a sensitive reporter mouse system

BACKGROUND: Targeted gene correction provides a potentially powerful method for gene therapy. RNA/DNA chimeric oligonucleotides were reported to be able to correct a point mutation with a high efficiency in cultured rodent cells, in the body of mice and rats, and in plants. The efficiency of correction in the liver of rats was claimed to be as high as 20% after tail-vein injection. However, several laboratories have failed to reproduce the high efficiency. METHODS: In order to sensitively detect and measure sequence changes by the chimeric oligonucleotides, we used Muta Mouse, a transgenic mouse system for mutation detection in vivo. It carries, on its chromosome, multiple copies of the lambda phage genome with the lacZ(+) gene. Two chimeric oligonucleotides were designed to make a point mutation at the active site of the LacZ gene product. They were injected into the liver with HVJ liposomes, which were demonstrated to allow reliable gene delivery. One week later, DNA was extracted from the liver, and lambda::lacZ particles were recovered by in vitro packaging. The lacZ-negative phage was detected by selection with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the injected mice was at the same level as the control mouse (approximately 1/10000). Our further restriction analysis and sequencing did not detect the designed mutations. CONCLUSIONS: Gene correction frequency in mouse liver by these oligonucleotides was shown to be less than 1/20000 in our assay with the Muta Mouse system.     

The novel dominant mutation Dspd leads to a severe spermiogenesis defect in mice

Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.