Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Low-multiplicity infection of Moloney murine leukemia virus in mouse cells: effect on number of viral DNA copies and virus production in producer cells.

Mouse cells infected with Moloney murine leukemia virus (M-MuLV) were prepared by two methods, and the number of M-MuLV-specific DNA copies in the infected cells was measured. The number of M-MuLV-specific DNA copies detected varied from one to eight per infected cell in different cell lines. Cells in which multiple rounds of viral infection occurred during establishment had on the average more viral DNA copies than cells in which infection at low multiplicity was performed, followed by cloning of the cells. However, even in cells derived by the low multiplicity of infection method, most cell lines carried more than one copy of M-MuLV-specific DNA. Virus production per cell was also measured, and no strict correlation was observed between the number of M-MuLV DNA copies present and the amount of virus produced.     

Transgene copy number-dependent rescue of murine beta-globin knockout mice carrying a 183 kb human beta-globin BAC genomic fragment

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.     

Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.     

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T₁ progeny from 26 T₀ plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.     

Cloning of Two Genetically Transmitted Moloney Leukemia Proviral Genomes: Correlation Between Biological Activity of the Cloned DNA and Viral Genome Activation in the Animal

The Mov-7 and Mov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the Mov-7 locus and Mov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the Mov-9 locus but is not activated in animals carrying the Mov-7 locus. Consequently, only Mov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the Mov-7 and Mov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the Mov-7 and Mov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10⁻⁵ XC plaques per genome equivalent were induced by the Mov-9 clone, whereas only 10⁻⁹ XC plaques per genome equivalent were induced by the Mov-7 clone. Moreover, NIH 3T3 cells transfected with the Mov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with the Mov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at the Mov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in Mov-7 and Mov-9 mice correlates with the biological properties of the respective clones. Genomic DNA from Mov-9 mice was not infectious in the transfection assay (specific infectivity < 10⁻⁷ PFU per genome equivalent). As the only difference between the genomic and the cloned Mov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.     

Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells

Sperm-mediated gene transfer in vertebrates has undergone various developments over the last few years, in different laboratories. In the present study, we microinjected a circular plasmid, carrying the lacZ reporter gene mixed with noncommercial cationic lipids, into the seminiferous tubules of anesthetized adult mice. Histochemical analysis was used to estimate the transfection efficiency 48-96 hr and 40 days after injection. As early as 48-96 hr post-injection, an efficient transfection was revealed by a beta-galactosidase expression within both immature and differentiated germ cells. By 40 days post-injection, the specific LacZ expression was restricted to the most immature germ cells in the basal portion of the seminiferous tubules. At this time, some injected males were mated with wild-type females and the progeny were analyzed by PCR and Southern blot. We showed that the transgene was transmitted to the offspring but remained episomal, as it was found in the tail of the young animals but not at adulthood. Therefore, the plasmid seemed to be lost during the numerous germ cells divisions. This plasmid stayed in some tissues, such as skeletal muscle and cardiac muscle. No integrative forms have yet been found with the use of a circular DNA.     

Altered expression after expansion of a v-erbA transgene in transgenic mice

Repetitive DNA is known to undergo size variations based on an increase or decrease in the number of monomer units. We describe here the spontaneous expansion of an experimentally introduced tandem array of repeats in a transgenic mouse consisting of the human -actin promoter fused to the viral oncogene v-erbA and an SV40 polyadenylation signal (hAP/v-erbA). The expansion of the transgene was identified during routine screening of transgenic offspring for heterozygosity or homozygosity in one founder line. The control heterozygote genome consisted of the hAP/v-erbA transgene organized as a tandem array approximately five monomer units at a single chromosomal locus. The number of units increased to 20–21 copies, and germline transmission of the expanded units was stable for at least two generations. The majority of animals carrying the expanded monomer units retained their RNA expression patterns; however, some of these animals had drastically reduced expression levels. We discuss the possibility that expansion of repeated units may provide a mechanism by which the expression of a deleterious transgene is reduced.     

Infectivity and structure of molecular clones obtained from two genetically transmitted Moloney leukemia proviral genomes.

The Mov-2 and Mov-10 substrains of mice, each carrying Moloney leukemia virus (= M-MuLV) in their germ line at the Mov-2 and Mov-10 locus, respectively, do occasionally at a later age (Mov-2) or not at all (Mov-10) activate infectious virus. The M-MuLV proviruses with flanking mouse sequences corresponding to the Mov-2 and Mov-10 locus, respectively, were molecularly cloned. Restriction enzyme analysis revealed no major deletions or insertions in the proviral genomes of the Mov-2 and Mov-10 locus. Both cloned DNAs induced XC plaques in a transfection assay. The specific infectivity, however, was very low and 3T3 cells transfected with the Mov-2 or Mov-10 clone did not produce infectious virus. Removing part of the 5' cellular sequences from the Mov-10 clone did not increase the infectivity. The results suggest that the M-MuLV integrated at the Mov-2 and Mov-10 locus carry a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of non-infectious particles.     

Spontaneous and bleomycin-induced genomic alterations in the progeny of Drosophila treated males depends on the Msh2 status. DNA fingerprinting analysis

Deficiency in DNA mismatch repair (MMR) confers instability of simple repeated sequences and increases susceptibility to cancer. Some of the MMR genes are also implicated in other repair and cellular processes related to DNA damage response. Supposedly, lack of their function can lead to a global genomic instability, besides microsatellite instability (MSI). To study the spontaneous and induced genomic instability in germ cells, related to the Msh2 status, DNA alterations in the progeny of individual crosses of Drosophila deficient in one or two copies of the Msh2 gene, were analysed by the arbitrarily primed polymerase chain reaction (AP-PCR). The results indicate that the progeny of homozygous parents for the normal Msh2 allele (+/+) presents a significantly lower frequency of genomic alterations than those from heterozygous (+/-) or mutant homozygous (-/-) parents. In addition, the DNA damage transmitted to the progeny, after the adult parental males were exposed to bleomycin, indicates that whereas the induction of mutations related to MSI depends on the lack of the Msh2 function, the induction of other mutational events may require at least one functional Msh2 allele. Thus, the results obtained with heterozygous individuals may have special relevance for cancer development since they show that a disrupted Msh2 allele is enough to generate genomic instability in germ cells, increasing the genomic damage in the progeny of heterozygous individuals. This effect is enhanced by mutagenic stress, such as occurs after bleomycin exposure.     

The effect of input DNA copy number on genotype call and characterising SNP markers in the humpback whale genome using a nanofluidic array

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.     

Perinatal lethality (ple): a mutation caused by integration of a transgene into distal mouse chromosome 15

We have used cytogenetic and recombinational analysis to determine the position of a transgene integrated into the mouse genome. The transgene maps to band F on the physical map of mouse chromosome 15 by in situ analysis and is tightly linked genetically to a cluster of loci that include the mutations caracul (Ca) and microcytic anemia (mk). Genetic analysis of the offspring of noninbred animals carrying the transgene and marker loci demonstrates a significant deficiency of homozygous progeny at weaning. When inbred mice heterozygous for the transgene are mated, about one-quarter of their offspring are homozygous; none of these animals survives more than 1 day after birth. It appears likely that a recessive insertional mutation has occurred as a result of transgene integration into a locus required for postnatal viability. We call this mutation transgenic perinatal lethality (Tg.ple).     

Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wld s ) gene

Background

The strain of MeCP2^tm1.1Bird mice is a broadly used model for Rett syndrome. Because males carrying the invalidated MeCP2 locus are sterile, this strain has to be maintained in a heterozygous state. All animals therefore have to be genotyped at every generation to discriminate those carrying the invalidated allele (+/- females and y/- males) from those that do not. This is conveniently carried out by PCR on tail genomic DNA but because the primer pairs described initially for this purpose yield very similar size DNA bands on the WT and the KO alleles, this requires to carry out two independent PCR reactions on tail DNA preparations from all animals.

Results

After cloning and sequencing the PCR fragment amplified on the KO allele, we tested several sets of primers that were designed to yield PCR fragments of different sizes on the KO and WT alleles.

Conclusion

We have thus identified a set of three primers that allows for efficient genotyping of the animals by a single PCR reaction. Furthermore, using of this set of primers also resolves a recurrent problem related to the tendency of one of the initial primers to give rise to a non specific band because of its capacity to anneal at both ends of a repeated genomic element which we have identified as MurvyLTR.     

Developmental fate of a human insulin gene in a transgenic mouse

Summary A plasmid containing a genomic human insulin clone was microinjected into a pronucleus of the fertilised mouse egg. Eggs were subsequently transferred into oviducts of pseudopregnant Swiss/Alb females. Embryos developed to term and the DNA was extracted from different organs. Southern blotting analyses revealed 1 transgenic female out of 96 animals born after microinjection of C57BL/6 mouse eggs. A tandem integration was found at one locus within the mouse genome and molecular rearrangement was found within this locus. The structure of the entire locus was identical in DNA from all tissues. Both the human insulin gene sequences and the pBR322 sequences were found to be extensively methylated, although some sites were hypomethylated in the pancreas and liver. The transgenic female produced ten offspring, none of which retained the insulin gene sequences. Seven offspring retained some pBR322 sequences which were stably transmitted to the F2 and F3 generations. Homozygous F3 pBR/pBR animals were obtained, which showed neither visible defects nor sterility. The loss of the tandem locus in the F1 generation did not seem to be due to mosaicism, but involved excision due to recombination. Sequences close to the ends of the tandem locus were involved in this event. A mechanism implying excision during germ cell formation is discussed.     

Evidence for the Tf locus being associated with an early lethal factor in a strain of pigs

The frequencies of three co-dominant alleles Tf ^A, Tf ^B and Tf ^c controlling the serum transferrin locus in native and exotic breeds of pig in the United Kingdom are shown. Matings between different transferrin genotypes and segregation of the transferrin alleles in piglets from 131 litters are also recorded.
In eleven litters from matings within a closely related group of animals heterozygous for the Tf ^c allele, no offspring of the Tf ^c/ Tf ^c genotype were born. Matings between heterozygous related boars believed to be carrying a lethal factor linked to the Tf locus and heterozygous unrelated sows of the Saddleback breed, resulted in offspring of the Tf ^c/ Tf ^c genotype being born.
An excess of heterozygote genotypes was found in all litters from matings between animals heterozygous for the Tf ^c allele. In all these litters the average litter size was 0.5 piglets less per litter than the average for all other litters studied. For the eleven litters from matings involving closely related animals heterozygous for the Tf ^c allele, the average litter size was 1.4 piglets less per litter than the average for all other litters.
The possibility of an early lethal factor being linked to the Tf locus in this family of pigs is discussed.     

An integrated Bayesian analysis of LOH and copy number data

Background  

Cancer and other disorders are due to genomic lesions. SNP-microarrays are able to measure simultaneously both genotype and copy number (CN) at several Single Nucleotide Polymorphisms (SNPs) along the genome. CN is defined as the number of DNA copies, and the normal is two, since we have two copies of each chromosome. The genotype of a SNP is the status given by the nucleotides (alleles) which are present on the two copies of DNA. It is defined homozygous or heterozygous if the two alleles are the same or if they differ, respectively. Loss of heterozygosity (LOH) is the loss of the heterozygous status due to genomic events.     

Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

Drosophila P element integration in the mouse

A recombinant plasmid containing the Drosophila melanogaster P element transposon was microinjected into mouse zygotes. Dot-blot analysis indicated that one of the newborns contained a single copy of the microinjected DNA per haploid mouse genome equivalent; two other newborns had integrated multiple copies of the P element construct. Southern mapping revealed that the entire plasmid, including both pBR322 sequences and P element sequences, had integrated in each of the three animals. In the two mice carrying multiple copies of the microinjected DNA, the copies appear to be linked in a tandem head-to-tail array. Therefore, in each of the three newborns integration of P element sequences has occurred by a mechanism which is distinct from that observed when the same plasmid is injected into Drosophila embryos. Analysis of DNA from the offspring of one of the transgenic mice showed no indication of transposition of P element sequences.