Occasionally I will come across articles that refer to back-crossing mice of one strain onto the background other another strain (e.g., C57Bl6). They do not explicitly state the purpose for doing so, but I can infer that the change of strain is to relate their findings to what is already known using the new strain (e.g., C57Bl6). Usually the back-crosses are performed for 5-10 generations. Is this to try to remove (epi)genetic variation from the original strain relative to the new strain, or is it more about normalizing expression of a particular allelic variant (e.g., knocking-in a transgene)?
Depending on the exact goal of the experiment, the researchers may back-cross both to smooth out genetic variation between individuals and to potentially normalize expression of a transgene, although once you get past the chimeric stage gene expression should be fairly stable. In my experience, back-crossing allows you to generate a genetically-altered mouse that differs as little as possible from a (generally well-characterized) parent strain, except at the locus of the genetic manipulation. This way, any differences in phenotype can more easily be explained by the knockout/knockin/transgene/what-have-you, and not by any variations in background genetics.
As an example, C57BL/6 mice, being inbred, have a number of well-known mutations, deficiencies, resistances, etc., and for one reason or another have become one of the most popular and most-characterized mouse strains in biology, so there is a great interest in continuing research using a well-known historical model with so much extant experimental data. These mice are quite different from the 129 strain, which are frequently used to make initial genetically modified strains due to the large number of embryonic stem cell lines available, and other technical reasons which allow for the relatively easy generation of transgenics, etc. However, for various other reasons, 129s aren't used as often in the type of studies that BL/6s are, so it's preferable to create the mutation in a 129, then back-cross it onto a BL/6 background. Both of these strains are quite homogenous from one individual to another (each sibling is practically a twin of the next), but once you start breeding 129s to BL/6s you can generate all sorts of genetic heterogeneity due to random cross-overs and all the other things we like to talk so much about around here when it comes to mutations and meiosis. As anyone who has worked with genetic diseases in humans will readily tell you, it can be extraordinarily difficult to nail down specific genotypes that lead to specific phenotypes when there are so many other confounding factors present. Backcrossing a mutant 5, 10, or even 20 or more generations back onto a reference strain is one of the best ways we have at the moment of controlling for all the genetic variations that are introduced when two inbred "tool" strains are bred together.
And now, cute pictures :)
both images from Charles River
What is the purpose of back-crossing mice for multiple generations? - Biology
The mouse is a popular model system because it is a mammal with sophisticated genetic tools and significant genetic resources.
Position among mammals
Mammalian orders arose from 50-65 million years ago, in a rapid diversification. The branching of the different mammalian orders was difficult to resolve on morphological and fossil evidence. Comparison of assembled whole genome sequences suggest that carnivores (dog) are more related to primates (humans) than rodents (mice).
Mice have 20 chromosomes in their haploid genome (thus 40 chromosomes in all). The haploid genome is about 3 picograms, similar to humans. The gene order of the genomes of mice and humans are conserved (synteny) although there are rearrangements, several per chromosome. Unlike the mostly metacentric chromosomes of humans, all mouse chromosomes are acrocentric.
Adult mice weigh 30-40 grams (50,000 to 70,000 grams for a young adult human) have a blood volume of 2 ml (4,800 ml for humans), and a resting heart rate of 500-700 bpm (60-80 bpm for humans).
Laboratory mice are unique in that there are a large number (hundreds) of inbred strains. Inbred strains are a strain of mice in which every individual is essentially genetically identical and homozygous at all loci.
Inbred strains of mice are generated by 20 generations or more of brother-sister mating. Although it is often stated that inbred strains of mice are homozygous at all loci, minor variations within an inbred strain have been found, and clear differences exist between the same strain maintained at different vendors or laboratories.
Most common inbred strains of mice arose from a limited number of genetically distinct mice, and thus the genome of many of the common inbred strains are a mosaic of small (several genes) contiguous stretches of one of a small number of genetic variants. Thus, for a span of several contiguous genes (300-600 kb), two strains may be essentially identical in DNA sequence (1 nucleotide change per 21,000 base pairs), or they may be divergent (1 nucleotide change per 440 base pairs). (Frazer et al., Nature 448:1050)
The advantages of inbred strains of mice include the fixation of genetic background and the reproducibility of that background in different laboratories and through time (for some strains, like the common C57BL/6J from The Jackson Laboratory, the strain has been archived as frozen embryos and the stock is replace from frozen embryos periodically). Given the mutation rate (1 x 10^-5 per locus per gamete), genetic drift is low, and all mice of a given strain are essentially genetically identical (excepting males/females)
When mice of two different inbred strains are mated, their offspring are said to be F1 (filial generation one) mice. F1 mice are genetically identical to each other, since each inherited the same paternal set of chromosomes and the same maternal set of chromosomes. F1 mice are more robust than their parents due to hybrid vigor. (Obviously, with the exception that males and females are chromosomally, genetically and phenotypically distinct. Also, there may be epigenetic parent-of-origin effects, such that F1's generated female strain1 x male strain2 could be different from F1's generated from female strain2 x male strain1--the two crosses in this example are called reciprocal crosses.)
F1 mice mated brother-sister produce an F2 generation. All F2 mice are genetically distinct from each other, since the paternally and maternally rederived chromosomes recombine before segregating into gametes.
An F1 mouse mated to its parent produces the first backcross generation, the N1 generation. Continued backcrosses to this strain generates subsequent backcross generations (N2, N3 etc).
The terms isogenic, coisogenic, congenic and consomic are used to describe specific types of relatedness. Isogenic mice are genetically identical, thus different individual mice of an inbred strain are isogenic. Coisogenic mice have a variant (mutation, transgene, targeted allele) which arose directly on that strain. Congenic mice have a variant larger than a gene but are otherwise isogenic. Congenic mice typically were mutants which arose in one strain, but which were backcrossed 9 generations or more onto a second strain, selecting for the desired variant at each generation. The generations are denoted like a backcross with N1, N2, etc. In a congenic, the variant as well as the contiguous genes (from the first strain) near the variant are brought into the background since recombination will not efficiently remove closely linked DNA. Consomic mice have a complete chromosome from one strain of mice in the background of a second strain. A set of 21 consomic lines can be used to represent each (nuclear) chromosome of one strain in a second.
There are also mice which are maintained as populations of genetically heterogeneous mice which are bred in rotation to maintain a high degree of heterozygosity. These strains are called outbred strains. Some outbred strains were put under selective pressure for high reproduction in their past. The robustness and high reproduction of the outbred strains make them useful in reproductive studies, reproductive technologies and in embryology, where large numbers of mutant embryos (which might be only 1/4, 1/8 or 1/16 of the embryos) are needed. The outbred strains of mice shouldn't be thought of as more representative of wild mice than inbred mice: both are laboratory artifacts, although generated by different breeding strategies and selective pressures.
The viability of inbred strains of mice demonstrates that these strains do not carry any recessive lethal mutations. Recessive lethal mutations and other deleterious mutations are present in all wild animal populations (it has been estimated that each individual carries 6 recessive mutations), thus inbreeding results in reduced fitness and lethality. It must be assumed that lethal and deleterious mutations were present in the progenitors of laboratory mice, but lethals weren't propagated and deleterious mutations were selected against in a laboratory setting. Inbred strains do have genetic defects characteristic of the strain, including agenesis of corpus callosum (129 strain), blindness (C3H, FVB, SJL) and progressive hearing loss (A/J, Balb/c, C57BL/6).
Mutations which affected the coat color of mice were among the first mutant alleles studied in the mouse, and they still are important today. Many inbred and outbred strains of mice carry a recessive mutant allele of a coat color gene (or combinations of recessive alleles). These recessive mutations were useful to detect contamination of an inbred strain with either wild mice, or another inbred strain.
It is useful to understand the genetics of two of the most common coat color genes, albino and nonagouti. These genes are names for their recessive mutant phenotypes, so mice homozygous mutant at the albino locus have no pigmentation (white fur and pink eyes) and heterozygotes and wild type for albino have pigmentation (dark fur and eyes). The albino gene product, tyrosinase, is necessary for the synthesis of all pigment. Mice have two different pigments in each hair, yellow at the tip and black in the rest of the hair. In albino mutants, neither pigment is made. The nonagouti gene product is necessary for the production of the yellow pigment, so mutant nonagouti mice have black hair. In nonagouti heterozygotes and wild types, the fur looks brown because of the combination of yellow and black pigments. Mice mutant for both albino and nonagouti are white.
Life Cycle and Reproduction
Human (and other higher primate) reproduction differs from the reproduction of most mammals, in that human reproduction is not seasonal and the ovulatory cycle is cryptic. Mouse reproduction in the wild varies seasonally, and the ovulatory cycle is evident and coordinates mating. Long daily light periods in the daily cycle support robust reproduction. Thus laboratory mice are maintained on a long (12 hours of light 12 hours of dark) light cycle.
The ovulatory or estrus cycle of female mice has a length of 4 to 6 days. The estrus cycle can become synchronized in female mice housed together continuously, and can be suspended in the absence of exposure to male pheromones. Suspended cycles can be restarted by exposure to male pheromones. Female mice are only receptive to mating when they have ovulated. Receptive females can be identified by inspection of the genitalia. The physical stimulation of the cervix in mating is required to make the uterus receptive to embryo implantation. The male produces a copulatory plug from secretions from the vesicular and coagulating glands that blocks the vulva for 8 to 10 hours after mating. Whether a female has mated can be determined by the plug, by inspection or with a blunt probe.
Estrus and ovulation can be manipulated by hormone injections to synchronize and maximize ovulation (superovulation). The early embryo (preimplantation embryo) is free in the oviduct and uterus of the female mouse for the first 4 days. Preimplantation embryos can be recovered from the oviduct and uterus by flushing culture media through the oviducts and uterine horns (the uterus of mice is bicornuate, having two tracts attached at the cervix.). Preimplantation embryos can be manipulated in culture by injection, infection and by adding or removing cells. They can be cultured and transferred surgically into a recipient female (embryo transfer). If the uterus of the recipient female is receptive, then pups can be born. Recipient females are mated to vasectomized males prior to the embryo transfer surgery to ensure that the uterus is receptive. These pseudopregnant females are mated according to a schedule such that the age of the transferred embryos is matched.
Gestation in the mouse takes 18 to 20 days, depending on the strain. Within a litter of embryos, there is some variation in the timing of development, which is particularly evident at early stages when morphological development is rapid.
The stages of embryos and fetuses are designated by a standardized nomenclature. This system expresses the stage of an embryo as the day of gestation. The midnight preceding the plug is designated as 0.0 days or E0.0. However, because individual embryos within a litter and embryos of different strains develop at different rates, the stage of an embryo, designated in days, is defined morphologically, in fact. For example, at eight days (midnight at the end of the eighth day), some embryos will have formed the first somite and will be E8.0, and others in a litter may be younger with well developed headfolds and be E7.75 and other might be older with 3 somites and be E8.25. Yes, it really is as confusing as you think it is.
Most litters which are born can be successfully reared by their mothers. Pups which are not viable may be partially or wholly consumed by mice in the cage. When the mother is not happy, however, she may eat the entire litter. A key component of keeping mice happy is to keep them in stable social groupings. Thus, male-female pairs, pairs of acquainted pregnant females, or a non-pregnant and pregnant female are all more likely to successfully rear offspring than individually housed females.
Mice are born hairless and with their eyes closed. By three weeks of age, they have their adult hair, open eyes, teeth and can jump to the top of the cage to feed and drink. At this time they can be removed from their mothers, and are typically housed together by sex.
For targeted and transgenic strains, mice carrying the DNA alteration are usually identified by taking a punch of tissue from the ear in a pattern to mark the animal and to provide DNA for a PCR assay (genotyping). Genotyping is most often performed at weaning.
Sexual maturity can occur as early as 6 weeks in females and 8 weeks in males, depending on strain.
The reproductive life span varies widely with strain, but females typically have a reproductive life span of 6-8 months, whereas males have a reproductive life span of about a year. In practice, it is important not to overestimate reproductive life span in order not to lose the ability to propagate the strain.
The life span of mice also varies with strain, but is 1.5-2 years typically.
Infectious Agents and Commensal Bacteria
In addition to standardized genetic background, much effort is put into standardizing the pathogen status of mice. It seems obvious that pathogens and cycles of infection could impact research results, but the standard of practice goes beyond pathogens with clinical effects to include some agents without clinical effects in normal mice. The rationale for elimination of subpathogenic agents is that they may have immunomodulatory effects, since mice are widely used for immunological research. This standard of care is called Specific Pathogen-Free. This standard has changed as new pathogens are discovered or their effects are better understood. For example, noroviruses and Helicobacter sp were added to the list of excluded pathogens recently.
Mice which have pathogens are made Specific Pathogen-Free by rederivation. Rederivation is an embryo transfer where the embryo donor mother is kept in quarantine, the embryos recovered sterilely and transported out of quarantine and transferred into a Specific Pathogen-Free pseudopregnant recipient female, housed in a facility which is Specific Pathogen-Free.
Commensal bacteria can affect physiology and interact with genetic variants in the host. In order to control the commensals, mice free of bacteria (axenic mice) can be generated by caesarian delivery. Axenic mice can then be inoculated with defined bacterial populations to generate gnotobiotic mice.
Primary cultures from mouse embryos or fetuses (Primary Mouse Embryonic Fibroblasts or MEFs) can be used to look at cellular phenotypes. MEFs can be immortalized by extended culture until they become transformed, or cell lines from specific tissues can be generated by expression of SV40 T antigen in the mouse in the tissue of interest.
Cell lines from preimplantation embryos can be generated. These embryonic stem cell lines (ES cells) can be used to introduce exogenous DNA and to alter endogenous DNA by gene targeting. These ES cells can be studied directly in culture or can be used to generate mice containing the genetic alterations. Existing mouse strains can also be used to generate new ES cell lines, also used for studying cellular phenotypes in vitro. ES cells have many advantages over MEFs, including more rapid proliferation and the formation of clonal colonies.
Preimplantation embryos can be manipulated to incorporate other cells, and if the added cells are embryo-like (for example ES cells), they will participate in the formation of an embryo, fetus, and live born animal. ES cells can participate in the formation of all adult tissues, including the germ line, the cells which produce gametes. These embryos, fetuses and adults which are mixtures of two genetically distinct cells are called chimeras.
Because the genomes of the different components of a chimera are in separate cells, the genomes do not recombine. Thus, if the host embryo is a/a and the ES cell is A/A, then both 'a' and 'A' sperm could be produced. But 'a' sperm must come from the host, and 'A' sperm must come from the ES cell. If this chimera is mated to a mouse which is a/a, then a/a offspring come from the host cells, and A/a offspring come from the ES cell.
In the preceding example, the a and A are symbols used for the nonagouti locus, where a/a mice are black, and A/a mice (and A/A mice) are brown. This scheme is the strategy most often used to determine if the ES cells transmitted through the germ line into offspring.
In a typical targeting experiment, only one of the two copies of the gene are targeted, so that only 50% of the brown offspring will carry the desired mutation.
ES cells are typically made from males. The host embryos are not selected for sex, so chimeras can be made with a male host embryo or a female host. Because of the dominant nature of testosterone, and the fact that it acts systemically, the male/female chimeras will be male mice.
Chimeras can also be used experimentally to examine the effect of a mutant gene on cells in the context of wild type tissue. This strategy can be used to determine if the effect of the mutant gene acts on the cell itself (autonomous) or on its adjacent or distant neighbors (nonautonomous).
Mice can be engineered to have an introduced gene, so-called transgenic mice. Typically, fertilized eggs (zygotes) are collected and microinjected with DNA. The DNA is injected into the sperm nucleus after fertilization but before it fuses with the egg nucleus--before fusion the haploid nuclei are called pronuclei, thus the injection is sometimes called pronuclear injection. Around 20% of the surviving injected eggs will have the DNA inserted at one site in a chromosome. The injected DNA typically inserts as multiple head-to-tail copies (10 to 100's of copies) at this one site.
The insertion of DNA by microinjection often (
10% of the time) results in mutation. This high rate of mutagenesis occurs not because 10% of the genome is functional, but because the insertion of DNA is often accompanied by deletion and rearrangement at the site of insertion.
Introduced genes most often are minigenes consisting of enhancer elements for tissue-specific expression, a transcriptional promoter, the coding sequences (cDNA) and a complete set of poly-adenylation signals.
Surprisingly, the expression level of a minigene transgene does not correlate with the number of copies.
The expression of transgenes can be lost in subsequent generations through epigenetic silencing, thus a means to monitor that the transgene continues to be expressed is important, and monitoring transmission of the DNA into offspring by itself is not enough.
The expression of transgenes can be altered by the chromosomal environment around them. These effects include level of expression, tissues expressed in, and propensity to be silenced.
Because not all transgenes are expressed as desired, and because the insertion may have disrupted an endogenous gene, multiple (
3) independent transgenic founder mice or lines should be analyzed to ensure that the phenotypes generated are due to the transgene. To facilitate this, each DNA-positive founder mouse from the injection is typically bred to wild type mice to establish a line of mice descending from that founder, and then the different founder lines are treated as independent experiments.
Systems for temporal control of transgene expression also exist. One of the most prevalent systems is the doxycycline/tetracycline system. The strategy involves control of gene expression using the administration of a drug. Typically, these systems involve two transgenes, one which expresses a transcription factor which binds to the drug which activates or inhibits the transcription factor the second has DNA sequences for binding of the transcription factor which regulate expression of the target sequences in cis.
The cloning and tiling of the genome with large (
200 kb) genomic fragments in bacterial artificial chromosome (BAC) libraries has made possible the use of these fragments as transgenes. Unlike minigene transgenics, these large fragments direct expression proportionate to their copy number in the genome, often in the expression pattern of the endogenous gene and they are much less susceptible to epigenetic silencing. In addition, technologies are available (e.g., recombineering) to modify the DNA in sophisticated ways.
The genes of ES cells in culture can be manipulated by homologous recombination. The ability to grow large number of cells permits the selection of cells with rare homologous recombination events, and the ability of ES cells to contribute to mice makes it possible to introduce these genetic alterations into mice, once the cells with rare events have been identified. The clonal growth of ES cells is important here again, in allowing the facile establishment of many individual lines of cells.
Most often, gene targeting is used to generate a mutation such that the gene no longer produces a functional product (a null allele of the gene). A null allele is the most important genetic tool for determining the normal function of a gene. These mutations are colloquially referred to as knockouts.
Gene targeting can used to introduce point mutations or other subtle sequence alterations or to put a complete new coding sequence into a gene. These mutations are colloquially referred to as knockins.
Because a gene may have multiple functions at different times and in different tissues, it is useful to be able to eliminate gene function at specific times or in specific tissues. These alleles are termed conditional alleles, or, colloquially, floxed (for flanked by loxP) alleles. Conditional alleles generated by homologous recombination have wild type function until exposed to Cre recombinase, which deletes a portion of the gene which was flanked by the DNA sequences which bind Cre, the loxP sequence. In cases where Cre coding sequences are fused to a mutant form of the ligand-binding domain of the estrogen receptor (Cre-ER), the drug tamoxifen can be administered to mice to activate Cre-ER, initiating a round of recombination.
Gene trap libraries of mutated genes in ES cells are an important genetic resource. Gene trap libraries are generated by the random insertion of gene trap vector DNA into genes, with subsequent molecular identification of the gene. Gene trap vectors are typically one of two types, splice acceptor traps or poly-A traps. In a splice acceptor trap, when the gene trap inserts into an intron in the correct orientation, the endogenous gene's splicing pattern is disrupted by splicing into the splice acceptor of the gene trap. Splicing into the gene trap also results in expression of a drug resistance gene, and the survival of such cells in the presence of the drug. In a poly-A trap, the gene trap vector has a promoter and coding sequences for a drug resistance gene, but no polyadenylation signals. In the absence of a polyadenylation signal, not enough of the drug resistance protein is made to allow survival. If a poly-A trap vector inserts upstream of the poly-adenylation signals of a gene in the correct orientation, then properly terminated and adenylated transcripts for the drug resistance gene will be made, along with the drug resistance protein. To disrupt function of the gene, poly-A traps also have a splice acceptor to disrupt splicing of the endogenous gene. The genes which have been trapped are identified by sequencing of cDNAs spliced into gene trap sequences. The collections of gene traps are now large enough that genes with introns have been trapped multiple times. Gene traps with insertions in introns toward the 5' end of the gene are more likely to result in complete loss of function (null).
Mutant Resources and Repositories
Scientific agencies world wide have recognized that their support is necessary for the preservation and distribution of variant mice. Mouse Genome Informatics (MGI) at The Jackson Laboratory maintains a database of mutant, transgenic and other variant mice which have been published. This is the place to start to determine which alleles of a gene have been published. MGI links to the International Mouse Strain Resource (IMSR), which is a database of mutants in public repositories as mice or cryopreserved gametes, embryos or ES cells. Alleles of genes in gene trap libraries are not currently accessible through MGI and IMSR. The two places to start your search for gene trap alleles are the International Genetrap Consortium and the Texas Institute for Genomic Medicine. Conditional gene traps also exist, and these can most easily be searched at the European Conditional Mouse Mutagenesis (EUCOMM) Program. Links to consortia working from the phenotypes of variants and induced mutants back to the mutated gene--forward or classical genetics--are available at MGI's Phenotypes and Mutants Community Resources page.
The Jackson Laboratory provides genomic DNA from many of their strains.
BAC libraries of genomic DNA have been made from a number of inbred mouse strains. Two of these libraries, from 129 and C57BL/6J, have been end-sequenced and tiled on the assembled genome, so that the contents of the clones are known. For 129 genomic clones, BACs containing your gene can be identified using the ensembl genome browser, selecting the DAS source "129S7/AB2.2 clones". If you wish to obtain a specific clone, clicking on the BAC will bring up a menu, and selecting the link at the bottom of the list will take you to the order form to purchase from the Sanger Institute. C57BL6J BAC clones can be identified with the UC Santa Cruz genome browser and purchased from BACPAC Resources Center CHORI. The locations of single nucleotide polymorphisms between these strains can be ascertained here on the Jax web site.
MBP Mouse colony management includes the following services.
- Setting up breeding pairs.
- Cage monitoring for pregnancies and newborn pups
- Record keeping of all essential data. Client colony data can be viewed live by access to colony data management system “MOSAIC”.
- Tail-cutting and ear-notching pups for genotyping as needed.
- Submission of mouse tissue for in-house genotyping or transfer to customer for testing.
- Weaning pups into holding cages.
- Communicating monthly colony reports to the customer.
- Monthly billing of UC account or customer PO for all vivarium and genotyping services.
All cages assigned to a particular client’s breeding project will be charged to the client’s account as a per diem (cost/cage/day). These include both breeding cages and cages of weaned pups.
When genotyping is necessary, pups will be ID'ed by toe-clipping (or ear-notching and tail-cutting). Pups will be identified by cage number, toe-clip (or ear-notch) number, and date of birth.
Mice imported from vendors like JAX, Harlan and CRL may be imported directly into our MBP conventional facility for custom breeding projects.
Mice imported from a customer’s own facility may require re-derivation by embryo transfer to enter the MBP conventional housing facility and will always require re-derivation to enter our SPF barrier housing. Descriptions of our MBP Clean Barrier (Conventional) housing and SPF barrier housing facilities can be found here Mouse Housing .
To explore how trauma affects generations of mice, researchers stressed mother mice. Their pups then exhibited both molecular and behavioral changes, such as taking more risks on an elevated maze. These changes persisted for up to five generations.
Mother separated from pups and traumatized. Mother often ignores pups.
Three-month-old male offspring mated with untraumatized females.
Offspring show epigenetic and behavioral changes without having experienced trauma.
Breeding carried out for six generations.
Epigenetic changes, such as methylation of DNA and alteration of RNA
Epidemiological studies of people have revealed similar patterns. One of the best-known cases is the Dutch hunger winter, a famine that gripped the Netherlands in the closing months of World War II. The children of women pregnant during the food shortages died earlier than peers born just before, and had higher rates of obesity, diabetes, and schizophrenia. Studies of other groups suggested the children of parents who had starved early in life—even in the womb—had more heart disease. And a look last year at historical records showed the sons of Civil War soldiers who had spent time as prisoners of war (POWs) were more likely to die early than the sons of their fellow veterans. (The researchers controlled for socioeconomic status and maternal health.)
But the human studies faced an obvious objection: The trauma could have been transmitted through parenting rather than epigenetics. Something about the POW experience, for example, might have made those veterans poor fathers, to the detriment of their sons' lives. The psychological impact of growing up with a parent who starved as a child or survived the Holocaust could itself be enough to shape a child's behavior. Answering that objection is where mouse models come in.
Mansuy began in 2001 by designing a mouse intervention that re-creates some aspects of childhood trauma. She separates mouse mothers from their pups at unpredictable intervals and further disrupts parenting by confining the mothers in tubes or dropping them in water, both stressful experiences for mice. When the mothers return to the cage and their pups, they're frantic and distracted. They often ignore the pups, compounding the stress of the separation on their offspring.
Mansuy says the mice's suffering has a purpose. "We're applying a paradigm that is inspired by human conditions," she says. "We're doing it to gain understanding for better child health."
Unsurprisingly, the pups of stressed mothers displayed altered behavior as adults. But to Mansuy's surprise, the behavioral changes persisted in the offspring's offspring. Initially, she thought this could be a result of the offspring's own behavior: Mice traumatized as pups could have been bad parents, replicating the neglect they experienced in childhood. Thus they might simply be passing on a behavioral legacy—the same lasting psychological effect that might explain such findings in humans.
To rule out that possibility, Mansuy studied only the male line, breeding untraumatized, "naïve" female mice with traumatized males, and then removing males from the mother's cage so that their behavior did not impact their offspring. After weaning, she raised the mice in mixed groups to prevent litter mates from reinforcing each other's behaviors.
Her lab repeated the procedure, sometimes going out six generations. "It worked immediately," she says of the protocol. "We could see that there were symptoms [in descendants] that were similar to the animals that were themselves separated." Descendants of stressed fathers displayed more risk-taking behavior, like exploring exposed areas of a platform suspended off the ground. When dropped in water, they "gave up" and stopped swimming sooner than control mice, an indicator of depressivelike behavior in mice.
Mansuy is "definitely a pioneer," says Romain Barrès, a molecular biologist at the University of Copenhagen. Other researchers have developed conceptually similar models, for example giving male mice altered diets or exposing them to nicotine and tracing metabolic and behavioral changes out for generations.
"If you're asking, ‘Does the experience of the parent influence the process of development?’ the answer is yes," says epigenetics researcher Michael Meaney at McGill University in Montreal, Canada, whose own studies have shown that differences in maternal care can have epigenetic effects on brain development. "Isabelle and others have documented the degree to which the experience of the parent can be passed on. The question [is] how."
Three massive freezers down the hall from Mansuy's office are filled with samples of mouse blood, liver, milk, microbiome, and other tissues. These serve as a −80°C archive of more than 10 years of data. Mansuy estimates she's collected behavioral data and tissue samples from thousands of mice altogether.
Isabelle Mansuy is searching for molecular changes that could explain how trauma in mice affects their offspring.
She hopes the biological markers of trauma are hidden in those freezers, waiting to be revealed. Many of the early mammalian epigenetics studies focused on DNA methylation, which "tags" DNA with methyl groups that switch genes off. But those changes seemed unlikely to be directly inherited: In mammals, methylation is mostly erased when egg and sperm come together to form an embryo.
Mansuy and others still think methylation could have some role. But they are also zeroing in on tiny information-rich molecules called small noncoding RNAs (sncRNAs). Most RNA is copied from DNA, and then acts as a messenger to instruct the cell's ribosomes to produce specific proteins. But cells also contain short strands of RNA that don't produce proteins. Instead, these noncoding RNAs piggyback on the messenger RNAs, interfering with or amplifying their function, thus causing more or less of certain proteins to be produced.
Mansuy and others think stress may influence sncRNAs, along with the many other biochemical changes it causes, from higher levels of hormones like cortisol to inflammation. They have focused on the sncRNAs in sperm, which may be especially vulnerable to stress during the weeks that newly formed sperm spend maturing in a twisting tube on top of the testes. Later, when sperm and egg come together, altered sncRNAs could modify the production of proteins at the very beginning of development in a way that ripples through the millions and millions of cell divisions that follow. "Hosts of signals happen as those cells become a zygote," says epigeneticist Tracy Bale at the University of Maryland in Baltimore. "If dad brings small noncoding RNAs that have an effect on mom's RNAs, that can change the trajectory of embryo development."
Bale found evidence that trauma can affect sncRNAs in sperm—and that the effects might be transmitted to offspring. She stressed mice during adolescence by barraging them for weeks at unpredictable intervals, with things like fox odors, loud noises, and bright light. Then, she examined the sncRNAs in their sperm and offspring. She found differences in nine types of sncRNAs, including one that regulates SIRT1, a gene that affects metabolism and cell growth.
She then created RNA molecules with similar alterations and injected them into early-stage embryos. When those embryos grew to adults, they carried RNA alterations like those seen in the sperm. This second generation also had lower levels of corticosterone, the mouse equivalent of cortisol, after a stressful spell inside a tight tube. "If you do the same RNA changes, you produce offspring with the same phenotype," Bale says.
Mansuy found similar RNA changes in her male mice traumatized as pups. They had higher levels of specific sncRNAs, including miR-375, which plays a role in stress response. Mansuy is convinced those molecular changes account for some of the inherited behavioral traits she documented. In one experiment, her team injected RNA from traumatized male sperm into the fertilized eggs of untraumatized parents and saw the same behavioral changes in the resulting mice.
But although the cause, in the form of altered RNA, and the effect, in the form of altered behavior and physiology, are identifiable in mouse experiments, everything else remains maddeningly difficult to untangle, especially in people. "The field has come a long way in the last 5 years," Bale says. "But we don't know what's going on in humans because we don't have a controlled environment."
Trauma to a mother mouse can alter the behavior of her descendants over multiple generations, like this father, son, and grandson.
Still, mouse data in hand, Mansuy has been looking for similar epigenetic changes in people. She analyzed blood samples from Dutch soldiers, collected before and after deployment to Afghanistan between 2005 and 2008. And she's working with clinicians in Nice, France, to examine blood samples from survivors of a horrific 2015 terror attack.
Other researchers had found altered sncRNAs in the blood of the soldiers. In 2017, for example, Dutch researchers showed soldiers exposed to combat trauma had recognizable differences in dozens of sncRNA groups, some of them correlated with PTSD. But Mansuy couldn't find the same kinds of RNA changes that appeared in her lab's mice. That could be because the soldiers' samples were years old, or simply because mice and people are different, showing the limits of mouse models. But Mansuy hopes it means epigenetic changes are sensitive to the type of trauma and when it occurs in the life course. Mice can never perfectly replicate human suffering, but, she says, "the best approach" for research "is to select a population of humans who have gone through conditions which are as similar as possible to our model."
That's where the Pakistani orphans come in. The children's chaotic early years may have some similarities to what the mice in Mansuy's lab experience, she says, including unpredictable separation from their mothers.
Early results are promising. "We have overlapping findings with the mouse model," Jawaid says. In a preprint uploaded last month to bioRxiv, Mansuy and Jawaid documented changes in the levels of fatty acids in the orphans' blood and saliva that mimicked changes in the traumatized mice—as well as similar sncRNA alterations. The presence of similar biomarkers "suggests that comparable pathways are operating after trauma in mice and children," Mansuy says.
In a conceptually similar effort to go from mice to people, biologist Larry Feig at Tufts University in Boston exposed male mice to social stress by routinely changing their cage mates. Their sperm had altered levels of specific sncRNA groups—albeit different ones from those altered in Mansuy's mice—and their offspring were more anxious and less sociable than the offspring of unstressed parents.
Working with a sperm bank, Feig then looked for the same sncRNAs in human sperm. He also asked donors to fill out the Adverse Childhood Experience (ACE) questionnaire, which asks about abusive or dysfunctional family history. The higher the men's ACE score, the more likely they were to have sperm sncRNA profiles matching what Feig had seen in mice.
But this body of research hasn't convinced everyone. Geneticist John Greally at the Albert Einstein College of Medicine in New York City has been a vocal critic of the evidence for epigenetic inheritance of trauma, pointing at small sample sizes and an overreliance on epidemiological studies. For now, he says, "Mouse models are the way to go." He's not yet seen definitive experiments even in mice, he says. "I'd like to see us be more bold and brave and move from preliminary association studies to definitive studies—and be open to the idea that there may be nothing there."
In a darkened room down the hall from Mansuy's office, just outside the mouse breeding area, two cages stand side by side on a table. One is a standard lab mouse enclosure, not much bigger than a shoebox. Wood chip–strewn cages like this are where most lab mice, including most of Mansuy's animals, spend their lives.
Next to it, black-furred, pink-tailed mice scurry up and down in a luxury two-story mouse house, equipped with three running wheels and a miniature maze. Their environment is designed to stimulate their senses and engage more of their brains in play and exploration.
In 2016, Mansuy published evidence that traumatized mice raised in this enriched environment didn't pass the symptoms of trauma to their offspring. The limited data—Mansuy says her lab is now working on an expanded study—suggest life experience can be healing as well as hurtful at the molecular level. "Environmental enrichment at the right time could eventually help correct some of the alterations which are induced by trauma," Mansuy says.
This and a few other studies suggesting epigenetic change is reversible have the potential to change the narrative of doom around the topic, researchers say. "If it's epigenetic, it's responsive to the environment," says Feig, who more than a decade ago found similar effects on brain function across generations by giving mice play tubes, running wheels, toys, and larger cages. "That means negative environmental effects are likely reversible."
In public talks and interviews, Mansuy says she's careful not to promise too much. As confident as she is in her mouse model, she says, there's lots more work to be done. "I don't think the field is moving too fast," Mansuy says. "I think it's moving too slow."
A mouse, plural mice, is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (Mus musculus). Mice are also popular as pets. In some places, certain kinds of field mice are locally common. They are known to invade homes for food and shelter.
Mice are classified under the order Rodentia. Typical mice are classified in the genus Mus.
Mice are typically distinguished from rats by their size. Generally, when a muroid rodent is discovered, its common name includes the term mouse if it is smaller, or rat if it is larger. The common terms rat and mouse are not taxonomically specific. Scientifically, the term mouse is not confined to members of Mus for example, but also applies to species from other genera such as the deer mouse, Peromyscus.
Domestic mice sold as pets often differ substantially in size from the common house mouse. This is attributable to breeding and different conditions in the wild. The best-known strain of mouse is the white lab mouse. It has more uniform traits that are appropriate to its use in research.
Cats, wild dogs, foxes, birds of prey, snakes and even certain kinds of arthropods have been known to prey heavily upon mice. Despite this, mice populations remain plentiful. Due to its remarkable adaptability to almost any environment, the mouse is one of the most successful mammalian genera living on Earth today.
In certain contexts, mice can be considered vermin. Vermin are a major source of crop damage,  as they are known to cause structural damage and spread disease. Mice spread disease through their feces and are often carriers of parasites.  In North America, breathing dust that has come in contact with mouse excrement has been linked to hantavirus, which may lead to hantavirus pulmonary syndrome (HPS).
Primarily nocturnal  animals, mice compensate for their poor eyesight with a keen sense of hearing. They depend on their sense of smell to locate food and avoid predators. 
In the wild, mice are known to build intricate burrows. These burrows have long entrances and are equipped with escape tunnels. In at least one species, the architectural design of a burrow is a genetic trait. 
Tissue-Specific Knockout Mice
While "housekeeping" genes are expressed in all types of cells at all stages of development, other genes are normally expressed in only certain types of cells when turned on by the appropriate signals (e.g. the arrival of a hormone).
To study such genes, one might expect that the methods described above would work. However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied.
Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell.
The Cre/loxP System
One of the bacteriophages that infects E. coli, called P1, produces an enzyme &mdash designated Cre &mdash that cuts its DNA into lengths suitable for packaging into fresh virus particles. Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP. All the DNA between the two loxP sites is removed, and the remaining DNA ligated together again (so the enzyme is a recombinase).
- the gene encoding Cre attached to a promoter that will be activated only when it is bound by the same transcription factors that turn on the other genes required for the unique function(s) of that type of cell
- a "target" gene, the one whose function is to be studied, flanked by loxP sequences.
- those cells that
- receive signals (e.g., the arrival of a hormone or cytokine)
- to turn on production of the transcription factors needed
- to activate the promoters of the genes whose products are needed by that particular kind of cell
The result: a mouse with a particular gene knocked out in only certain cells.
5 Coisogenic, Congenic, and Segregating Inbred Strains
There are several ways in which inbred strains may differ at only a small part of the genome.
5.1 Coisogenic Strains
Coisogenic strains are inbred strains that differ at only a single locus through mutation occurring in that strain. Strains containing targeted mutations in ES cells that are then crossed to, and maintained, on the same inbred substrain from which the ES cells were derived can be regarded as coisogenic, but the possibility of mutations elsewhere should be considered. Similarly, chemically or radiation induced mutants on an inbred background can be considered coisogenic, although other genomic alterations could be present. A coisogenic strain may accumulate genetic differences over time by genetic drift unless periodically backcrossed to the parental strain.
Coisogenic strains should be designated by the strain symbol (and where appropriate the substrain symbol) followed by a hyphen and the gene symbol of the differential allele, in italics.
129S7/SvEvBrd-Fyn tm1Sor A targeted mutation of the Fyn gene was produced using the AB1 ES cell line derived from 129S7/SvEvBrd. Chimeric animals were mated to 129S7/SvEvBrd and the allele subsequently maintained on this coisogenic strain.
In some cases, such mutations will be maintained in heterozygous condition. It should be noted that this means that the strain designation does not reflect the breeding system, nor indicate the specific genotype of a given mouse or rat.
C57BL/6J-Aqp2 cph The congenital progressive hydronephrosis mutation in the aquaporin 2 gene arose on the C57BL/6J strain. It is a coisogenic strain, but because homozygotes are generally juvenile lethal, the strain is maintained by breeding heterozygotes Aqp2 cph /+ x Aqp2 cph /+.
If the number of generations of inbreeding since the mutation arose in a coisogenic strain is to be shown, it can be indicated by adding the number of generations since the mutation to the number before:
F110 + F23 indicates 23 generations of brother x sister matings since the occurrence of a mutation at F110 in an inbred strain.
5.2 Congenic Strains
Congenic strains are produced by repeated backcrosses to an inbred (background) strain, with selection for a particular marker from the donor strain (Snell 1978, Flaherty 1981). Congenic lines that differ at a histocompatibility locus and therefore resist each other's grafts are called congenic resistant (CR) lines.
A strain developed by this method is regarded as congenic when a minimum of 10 backcross generations to the background strain have been made, counting the first hybrid or F1 generation as generation 1. At this point the residual amount of unlinked donor genome in the strain is likely to be less than 0.01. (Note that the amount of donor genome linked to the selected gene or marker is reduced at a much slower rate, approximately equivalent to 200/N, where N is the number of backcross generations for N>5 (Flaherty 1981 Silver 1995).
Marker assisted breeding or marker assisted selection breeding, also known as "speed congenics" permits the production of congenic strains equivalent to 10 backcross generations in as few as 5 generations. (Markel et al. , 1997 Wakeland et al ., 1997). Provided that the appropriate marker selection has been used, these are termed congenic strains if the donor strain contribution unlinked to the selected locus or chromosomal region is less than 0.01. Ideally, descriptions of speed congenic strains in first publications thereof should include the number and genomic spacing of markers used to define the congenicity of the strain. Because speed congenics depend upon thorough marker analysis and can vary by particular experimental protocol, the inbred status of speed congenics should be regarded with caution.
Congenic strains are designated by a symbol consisting of three parts. The full or abbreviated symbol of the recipient strain is separated by a period from an abbreviated symbol of the donor strain, this being the strain in which the allele or mutation originated, which may or may not be its immediate source in constructing the congenic strain. A hyphen then separates the strain name from the symbol (in italics) of the differential allele(s) introgressed from the donor strain.
In cases where the chromosome on which the mutation arose is unknown, e.g. , the donor is not inbred or is complex or an F1 hybrid, the symbol Cg should be used to denote this complex genetic origin. The use of the donor strain symbol or Cg is essential to distinguish congenic from coisogenic strains. Cg also is used to designate a strain constructed by crossing together two congenic strains that have been backcrossed separately to the same host background, but where their respective donor strains differ. Cg also is applied where alleles originate from a single donor strain, but the congenic strain also carries other coisogenic alleles. The use of Cg after the period as the donor strain indicates that multiple alleles in the strain name came from more than one source or that the genetic origin of at least one allele in the strain name is uncertain.
B6.AKR-H2 k A mouse strain with the genetic background of C57BL/6 but which differs from that strain by the introduction of a differential allele (H2 k ) derived from strain AKR/J. SHR.BN-RT1 n A rat strain with the genetic background of SHR but which differs from that strain by the introduction of a differential segment (RT1 n ) derived from strain BN. ACI.BUF-Pur1/Mna A rat strain with the genetic background of ACI onto which a segment from the BUF strain containing the Pur1 QTL has been transferred. B6.Cg- Kit W-44J Gpi1 a A mouse strain with the genetic background of C57BL/6, but where the donor strain is mixed, the Kit allele originating from C3H/HeJ and the Gpi1 allele originating from CAST/Ei.
If several lines derived from the same host background and donor strains and carrying the same differential allele(s) are available, the individual lines should be distinguished by adding a forward slash followed by serial numbers and Laboratory codes.
Parentheses may be used to show that an inbred, incipient congenic or congenic inbred strain may have a minor contribution from other than the defined host background and donor strain. A single additional strain contribution is indicated by the strain abbreviation in parentheses following the inbred or congenic designation. Multiple, mixed or unknown additional contributions are indicated by the symbol Cg in parentheses. If the donor is designated by Cg, parenthetical information may not be included.
B6(C)-mut A mutation originates on an inbred ( e.g. , C57BL/6J), is crossed out to or onto another background (e.g., BALB/c) and then is crossed back onto the original background. B6(Cg)-mut A mutation arose in C57BL/6, was crossed onto a mixed or undefined background, and then was backcrossed back onto the original C57BL/6 background. C.129P(B6)-Il2 tm1Hor A targeted mutation created in a 129 ES cell line and transferred from a B6129P mixed background to BALB/c. B6(C)-mut A mutation arises on a congenic strain carrying another mutation (e.g., B6.C-m) and the original mutation is bred out of the new strain. The new mutation is known to have occurred in a host strain-derived segment of the genome. B6(C)-mut A mutation arises on a hybrid or mixed background stock (e.g., B6CF1) and is backcrossed onto one of the original inbred strains (e.g., C57BL/6J). The new mutation is known to have occurred in a host strain-derived segment of the genome. B6.C(Cg)-mut A mutation arose on an inbred strain (e.g., BALB/c) and was maintained on a mixed or undefined background (e.g., a linkage-testing stock) before being backcrossed onto a second inbred strain (e.g., C57BL/6).
If the chromosomal segment that has been transferred is defined by several genes or multiple DNA loci, the segment can be defined in the symbol by listing the most proximal and the most distal markers demonstrated to be in the segment in parentheses, separated by a hyphen.
B6.Cg-(D4Mit25-D4Mit80)/Lt A congenic strain made by introducing into C57BL/6J a segment of chromosome 4 from an outbred or mixed strain (=Cg), extending between the two defined markers. B6.CBA-(D4Mit25-D4Mit80)/Lt A similar congenic strain in which the donor chromosomal segment comes from the CBA/J strain.
Note that the markers defining the segment only describe the most proximal and distal markers tested, and this does not imply that there are not other untested markers further proximal or distal. If several lines are made, in the same or different labs that contain the same segment and would be otherwise indistinguishable, then a forward slash, serial number and Laboratory code should be appended.
If necessary, the number of backcross generations should be indicated by N and the number in parentheses following the strain name generations should not be incorporated into the strain name. Incipient congenics may be given congenic nomenclature at N5, as long as the number of generations of backcrossing is clearly documented in information accompanying the strain. In cases where it is necessary to use more complex mating systems, the generations should be expressed as N equivalents (NE) and the strain regarded as congenic at a minimum of NE10. For example, when backcrossing a recessive gene onto an inbred background, after 10 rounds of backcrossing and intercrossing to recover a homozygote for the next backcross (20 generations), the strain would be at NE10. When a congenic strain is maintained by brother x sister matings after backcrossing, the number of brother x sister generations follows the number of backcross generations, e.g. , (N10F6), 10 generations of backcrossing followed by 6 generations of brother x sister inbreeding (NE12F17), a complex system of backcrosses and intercrosses genetically equivalent to 12 backcrosses, followed by 17 generations of brother x sister matings.
When generating speed congenics N will be less than 10 initially, nevertheless the actual number should be given in parentheses following the N, e.g. , N(6), and the details of breeding system and markers used detailed elsewhere in a publication or database.
5.3 Chromosome Substitution or Consomic Strains
Chromosome substitution or consomic strains (Nadeau et al ., 2000) are produced by repeated backcrossing of a whole chromosome or its parts onto an inbred strain. The term chromosome substitution strain is a common designation for consomic, subconsomic, and conplastic strains. To create a chromosome substitution strain, transfer of a whole chromosome or a large chromosomal region is carried out, while in congenic strains, the transferred entity is a gene, marker or genomic segment including a specific marker or interval.
5.3.1 Consomic Strains
Consomic strains are produced by repeated backcrossing of a whole chromosome onto an inbred strain. As with congenic strains, a minimum of 10 backcross generations is required, counting the F1 generation as generation 1. For autosomes it is necessary to genotype progeny to ensure that the selected donor chromosome has not recombined with the corresponding recipient chromosome. The generic designation for consomic strains is HOST STRAIN-Chr # DONOR STRAIN .
SHR-Chr Y BN In this consomic rat strain, the Y chromosome from BN has been backcrossed onto SHR. C57BL/6J-Chr 19 SPR In this consomic mouse strain, a M.spretus Chromosome 19 has been backcrossed onto C57BL/6J. C57BL/6J-Chr 1 A/J Chr 3 DBA/2J In this consomic mouse strain, Chromosome 1 from the A/J strain and Chromosome 3 from the DBA/2J strain have been backcrossed onto C57BL/6J.
Experience shows that on occasion it is impossible to transfer an entire chromosome from one strain to another due to lethal effects on a particular chromosome. For example, a consomic set on which PWD/Ph individual chromosomes were transferred to C57BL/6J revealed that Chr 11 and Chr X cannot be transferred intact. To designate "sections" of transferred chromosomes that contribute to a consomic set, regions can be indicated as a decimal 1, 2, 3, etc.
Thus, a part of Chr 11 of this consomic set would be: C57BL/6J-Chr 11.1 PWD/Ph /ForeJ.
Although consomic strains are similar in concept and development to congenic strains, in consomic nomenclature the name of the host strain is not abbreviated, and no period followed by the donor strain is required because the strain of origin is shown in the superscript. Capitalization of all letters in the superscript and non-italicization of the chromosome letter/number and of the superscript distinguish a chromosome identifier from an allele symbol.
5.4 Segregating Inbred Strains
Segregating inbred strains are inbred stains in which a particular allele or mutation is maintained in heterozygous state. They are developed by inbreeding (usually brother x sister mating) but with heterozygosity selected at each generation. They are designated like other inbred strains since the segregating locus is part of the standard genotype of the strain (see Section 5.1 Coisogenic Strains). When segregating coat color alleles are part of the inbred strain's normal phenotype, they need not be included in the strain name (see examples below). Details of inbred strain genotypes are available in publications and databases.
129P3/J This mouse strain segregates for the tyrosinase alleles albino (Tyr c ) and chinchilla (Tyr c-ch ). WB/Re This mouse strain segregates for the dominant white spotting allele of the kit oncogene (Kit W ).
Strains that carry linked alleles in coupling or repulsion should be designated so that it is clear that the alleles are linked and the phase of the linked genes is specified.
B6.Cg-m Lepr db /+ + In this strain the m andLepr db alleles are carried on one chromosome (in coupling) and the wild type alleles on the other. B6.Cg-m +/+ Lepr db In this strain the m and Lepr db alleles are carried on different homologs of the chromosome (in repulsion) this is also called a balanced strain.
5.5 Conplastic Strains
Conplastic strains are strains in which the nuclear genome from one strain has been introduced into the cytoplasm of another, either by backcrossing (in which the mitochondrial donor is always the female parent) or by direct nuclear transfer into an enucleated zygote. The designation is NUCLEAR GENOME-mt CYTOPLASMIC GENOME .
C57BL/6J-mt BALB/c A strain with the nuclear genome of C57BL/6J and the cytoplasmic (mitochondrial) genome of BALB/c.
Such a strain is developed by crossing male C57BL/6J mice with BALB/c females, followed by repeated backcrossing of female offspring to male C57BL/6J. As with congenic strains, a minimum of 10 backcross generations is required, counting the F1 generation as generation 1.
Researchers succeed in making generations of mouse clones
Mouse clones from the 24th and 25th generations. Credit: RIKEN
Using the technique that created Dolly the sheep, researchers from the RIKEN Center for Developmental Biology in Kobe, Japan have identified a way to produce healthy mouse clones that live a normal lifespan and can be sequentially cloned indefinitely.
Their study is published today in the journal Cell Stem Cell.
In an experiment that started in 2005, the team led by Dr. Teruhiko Wakayama has used a technique called somatic cell nuclear transfer (SNCT) to produce 581 clones of one original 'donor' mouse, through 25 consecutive rounds of cloning.
SNCT is a widely used cloning technique whereby a cell nucleus containing the genetic information of the individual to be cloned is inserted into a living egg that has had its own nucleus removed. It has been used successfully in laboratory animals as well as farm animals.
However, until now, scientists hadn't been able to overcome the limitations of SNCT that resulted in low success rates and restricted the number of times mammals could be recloned. Attempts at recloning cats, pigs and mice more than two to six times had failed.
"One possible explanation for this limit on the number of recloning attempts is an accumulation of genetic or epigenetic abnormalities over successive generations" explains Dr. Wakayama.
To prevent possible epigenetic changes, or modifications to DNA function that do not involve a change in the DNA itself, Wakayama and his team added trichostatin, a histone deacetylase inhibitor, to the cell culture medium. Using this technique, they increased cloning efficiency by up to 6-fold.
By improving each step of the SCNT procedure, they were able to clone the mice repeatedly 25 times without seeing a reduction in the success rate. The 581 healthy mice obtained in this way were all fertile, they gave birth to healthy pups and lived a normal lifespan of about two years, similar to normally conceived mice.
"Our results show that there were no accumulations of epigenetic or genetic abnormalities in the mice, even after repeated cloning," conclude the authors.
Dr. Wakayama adds, "This technique could be very useful for the large-scale production of superior-quality animals, for farming or conservation purposes."
Dr. Wakayama's work made the news in 2008 when his team created clones from the bodies of mice that had been frozen for 16 years, using SNCT.
Sharing Laboratory Resources: Genetically Altered Mice: Summary of a Workshop Held at the National Academy of Sciences, March 23-24, 1993.
“Important as transgenic mice are, they are really the tip of the iceberg when compared with what we are going to see in the next few years.”–Janet Rossant
Several hundred mouse stocks containing mutations have been used for a long time as models of human disease and for the study of metabolic processes. Traditionally, the mutations have been spontaneous or induced by chemicals or radiation. Recently, scientists have gained the ability to create mutant strains by integrating foreign DNA, with increasing specificity, into the genome of mouse cells. Such transgenes can be added at random to the genome. Offspring of the resulting mouse express the trait coded for by the foreign DNA. Alternatively, mutations can be targeted at specific loci to produce “knockout mice” which do not express the gene, or otherwise to alter gene expression. Those approaches are now revolutionizing genetic research.
C57BL/6J is the most widely used inbred strain. It is commonly used as a general purpose strain and background strain for the generation of congenics carrying both spontaneous and induced mutations. Although this strain is refractory to many tumors, it is a permissive background for maximal expression of most mutations. C57BL/6J mice are used in a wide variety of research areas including cardiovascular biology, developmental biology, diabetes and obesity, genetics, immunology, neurobiology, and sensorineural research. C57BL/6J mice are also commonly used in the production of transgenic mice. Overall, C57BL/6J mice breed well, are long-lived, and have a low susceptibility to tumors. Primitive hematopoietic stem cells from C57BL/6J mice show greatly delayed senescence relative to BALB/c and DBA/2J. This is a dominant trait. Other characteristics include: 1) a high susceptibility to diet-induced obesity, type 2 diabetes, and atherosclerosis 2) a high incidence of microphthalmia and other associated eye abnormalities 3) resistance to audiogenic seizures 4) low bone density 5) hereditary hydrocephalus (early reports indicate 1 - 4 %) 6) portosystemic shunts (
5%) 7) hairloss associated with overgrooming 8) a preference for alcohol and morphine 9) late-onset hearing loss 10) increased incidence of hydrocephalus and malocclusion and 11) spontaneous calcaneal luxation in 1% of aged males beginning at 6-8 months of age, resulting in ankylosing enthesopathy of that tarsal joint.
C57BL/6J mice fed a high-fat diet develop obesity, mild to moderate hyperglycemia, and hyperinsulinemia (see JAX® Diet-induced Obesity (DIO) Models). C57BL/6J mice fed an atherogenic diet (1.25% cholesterol, 0.5% cholic acid and 15% fat) for 14 weeks develop lesions in the range of 4500 to 8000 um 2 atherosclerotic aortic lesions/aortic cross-section. The variation in aortic lesions found among various inbred strains has led to the identification of the existence of eight genes affecting atherosclerosis, Ath1 to Ath8. C57BL/6J mice also develop severe and progressive hearing loss later in life, with the disruption of both outer and inner hair cells, due to the Cdh23 Ahl allele. Cheers and McKenzie found C57BL/6J resistant to listeriosis. A naturally occurring deletion in nicotinamide nucleotide transhydrogenase (Nnt) exons 7-11 occurred in C57BL/6J sometime prior to 1984. This deletion results in the absence of the NNT protein, and is associated with impaired glucose homeostasis control and reduced insulin secretion. This mutation is not found in C57BL/6JEi, C57BL/6N, C57BL/6NJ, C57BL/6ByJ, C57BL/10J, C57L/J, or C58/J (Toye AA, et al, Diabetologia, 2005). Since C57BL/6JEi separated from C57BL/6J in 1976, the Nnt deletion arose sometime between 1976 and 1984. The spontaneous n-Tr20 m1J C50T point mutation, which is also present in C57BL/6JEiJ but not C57BL/6NJ or C57BL/6ByJ, causes increased ribosomal pausing at AGA codons compared with that of other inbred strains (Ishimura et al., 2014). n-Tr20 has been found to be restricted in expression to the nervous system and n-Tr20 m1J causes changes in synaptic transmission and raises the electroconvulsive seizure threshold, making C57BL/6J comparatively seizure resistant (Kapur et al., 2020). Additionally, an intronic point deletion in Gabra2, which arose sometime between the early 1970's and 1990's, results in decreased transcript and protein expression of this chloride channel component in the brain.
C57BL/6J was the DNA source for the international collaboration that generated the first high quality draft sequence of the mouse genome. 5 SNP differences have been identified that distinguish C57BL/6J from C57BL/6ByJ and C57BL/6NJ. Both C57BL/6ByJ and C57BL/6NJ type as follows: 08-015199792-M (rs3709624) is C 11-004367508-M (rs3659787) is A 13-041017317-M (rs3722313) is C 15-057561875-M (rs3702158) is G 19-049914266-M (rs3724876) is T. C57BL/6J types as follows: 08-015199792-M (rs3709624) is T 11-004367508-M (rs3659787) is G 13-041017317-M (rs3722313) is T 15-057561875-M (rs3702158) is A 19-049914266-M (rs3724876) is G (Petkov and Wiles, 2004.) Others have subsequently identified further SNP differences between sublines of C57BL/6 (Mekada et al., 2009, Zurita et al., 2010).
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