Mutants Polymorphisms Transgenic and Gene Targeted Mutations Collectively Known as Mutants

Several thousand mutants, polymorphisms, and transgenes of various sorts are now used in biomedical research, and the numbers will increase substantially over the next few years as targeted mutations and mutants produced by chemical mutagenesis continue to be produced. These have a Mendelian mode of inheritance, though there may be complications due to variation in gene expression, penetrance, viability, and breeding performance. The spontaneous or induced mutations and polymorphisms may be caused by alteration of a single base pair, which may cause a mutation in a coding or regulatory region or be genetically silent, or by a deletion or insertion of a larger tract of DNA. In some cases, such as the dilute coat color allele in DBA/2 mice, the mutation has been caused by the insertion of a retrovirus within the gene.56

Transgenic (including knockout) strains are produced by incorporation of foreign DNA into the genome as a result of direct injection into early embryos or via embryonic stem cells. Such DNA is incorporated into the host chromosomes, and with the right regulatory sequences, it can be transcribed and translated by the host as though it was a host gene. So-called knockout mice are produced by inactivating an existing gene. Briefly, once a gene has been sequenced, an inactive copy of it can be synthesized in vitro, usually by splicing a gene for resistance to neomycin in the middle of the sequence. This construct is then incorporated into embryonic stem cell cultures in vitro. When these cells are cultured, a small amount of homologous recombination occurs, with the inactive gene replacing the host gene. Clones of cells in which this has occurred are identified by selective markers. These cells are then injected into blastocyst stage embryos, usually from an unrelated strain, and returned to a foster mother. Some of the resulting offspring are chimeras composed of cells from the host blastocyst and the cloned cells. Coat color markers are usually used so that chimeric offspring can easily be identified. In some cases, the gonads of the chimeric animals will also be chimeric, in which case offspring can be produced whose genomes contain the knocked-out gene. An overview of mutagenesis and transgenesis is given by Silver,2 and more detailed descriptions of methods of producing transgenic animals of various species is given by Maclean.57 The techniques are advancing rapidly and include the possibility of site- and time-specific mutagenesis by knocking out a gene only in a particular tissue at a time chosen by the scientist, and also "knock-in" methods for replacing a gene with an alternative, say from another species.

Transgenic strains are used in a number of ways:

1. They can be used to produce valuable proteins, in milk or other body fluids, that would otherwise be difficult to synthesize or extract. For example, transgenic sheep are currently being used to produce human alpha-1-antitrypsin, which will be used to treat patients with cystic fibrosis and emphysema.58

2. They can be used to produce animal models of human disease or to study the effects of environmental agents. For example, several strains of mice that are highly susceptible to cancer are being evaluated in the hope that through them, we will be better able to detect carcinogenic chemicals that would be a hazard to humans and the environment.59

The major use of these animals will be in studying the functions of genes. There appear to be about 30,000 genes in humans and laboratory mammals, and the function of only a few thousand of them is known. Finding the function of the remainder, and using the knowledge to combat human disease, are the challenges now facing biomedical research.

Genetic Background of Mutants and Transgenes

Possibly the most important general point about this class of stock is that where a mutation causes a phenotype, this is virtually always subject to modification by other genes. A mutation that causes death or severe disability on one genetic background may be viable and have mild to nonexistent effects on another. Where a mutant or transgene is on a genetically segregating background, it is nearly always good research practice to backcross to one or more isogenic backgrounds before starting detailed investigation of the phenotype.60 With an outbred background, genetic drift and the effects of natural selection for reduced expression of deleterious phenotypes in the first few generations may mean that results are not constant over a period of time.

Characteristics and Research Uses of Mutants and Polymorphisms

Mutants may have occurred spontaneously or been induced by irradiation or chemicals. In the mouse, such mutants have been preserved for many years. Most mutants have some deleterious effect in the homozygous or heterozygous state, in contrast with polymorphisms discussed below. Many mutants have been identified as animal models of similar mutations in humans, while others such as the athymic nude mice and rats have also been widely used, because they have characteristics that are useful in a wide range of different disciplines. With the development of new molecular tools for studying mutants, it is now being realized that mutants who at first sight appear to be of little biomedical interest, such as coat color, circling, or curly tail mutants, often alter quite basic biological processes. Thus, they are a valuable resource for fundamental studies.

One of the advantages of mutants in research is that they are usually viable, and in many cases, there are several different mutant alleles at each locus, often with various gradations of severity. In some cases, this makes them more useful than knockouts. However, the obvious disadvantage is that if there are no mutations that affect a system of interest to a particular investigator, the only option may be to make an artificial one using transgenic methods. For people interested in basic research, a good strategy is often to find a new mutant, map it, identify the gene at the molecular level, and work out its mode of action. Many such studies are now reported in the literature, and more can be expected in the future. Although these studies will not lead to an immediate cure for human disease, in the long term, the knowledge accumulated in this way certainly will be useful.

Polymorphisms are genetic variants that are genetically silent or are present at a high frequency in a population. For example, several enzyme loci such as glucose phosphate isomerase (Gpil) are polymorphic, with the alleles being recognizably following electrophoresis and appropriate staining. Inbred strains can differ at this locus. Similarly, there is extensive polymorphism at some repetitive loci. Microsatellite loci, which are short simple repetitive sequences of base pairs such as CA, repeated about 50 to 200 times are highly polymorphic in most species and are widely used as genetic markers for mapping and genetic quality control.2 Minisatellite loci are also highly polymorphic, probably due to a high mutation rate.61

Recently, it has been found that there is extensive polymorphism of single nucleotides throughout the genome. These may occur within coding loci in which case they may or may not cause a mutation in introns and regulatory sequences where they may affect gene function, and in various types of noncoding DNA. These single nucleotide repeats (SNPs) are potentially valuable genetic markers once they are fully mapped and characterized,62 as well as being of biological importance if they alter gene regulation or transcription.

Nomenclature of Mutants and Polymorphisms

The nomenclature of mutants is reasonably straightforward. However, there are some complications, because gene symbols are changed frequently as a result of genetic advances. Thus, mutants such as obese were given a symbol, in this case ob, which indicated the allele and the locus. The wild type at the ob locus was then designated +ob or, when the context was clear, just +. However, when the gene was mapped and cloned, it was found to code for a protein named leptin, which was given the locus symbol, Lep, so the obese allele has now been renamed Lep°b. Now the wild type is designated +Lep or Lep+. Many mutants are undergoing such changes in their designations, with old symbols such as c for the albino locus now being redesignated Tyrc. The nude mutation has been redesignated Foxn1nu, because it is a mutation at a locus first described in Drosophila, which causes a forked head in that insect. As far as possible gene symbols for loci that are recognizable as identical in different species should be uniform across species.

Full details of the genetic nomenclature rules for mice are given on the Jackson Laboratory Web site (www.informatics.jax.org). In short, names for genes, loci, and alleles should be brief and, if possible, descriptive, e.g., "obese" or "congenital hydrocephalus." Genes are functional units, whereas a locus can be any distinct DNA sequence. Symbols for genes should be short abbreviations of one to four letters, starting with the same letter as the name. Arabic numbers can be included as part of the name where necessary, but the first symbol should always be a letter. Roman numerals and Greek letters should not be used. Hyphens are only used for clarity, such as when two numbers need to be separated. In published articles, gene symbols are given in italics.

Loci defined by anonymous DNA probes are given a symbol starting with a D followed by the chromosome assignment (i.e., the numbers 1 to 19, X or Y), a laboratory registration code (see above for nomenclature of inbred mouse strains), and a unique serial number. The laboratory methods for detection of the locus also need to be specified. Alleles are usually designated by a superscript. Where this is not possible (e.g., where superscripts are not accepted electronically), the symbol can be enclosed in chevrons, e.g., Gpi1a or Gpi1<a>.

There are additional rules relating to things like pseudogenes, super-gene families or complexes, retroviruses, and for special classes of genes and gene complexes such as biochemical variants, lymphocyte antigens, histocompatibility loci, etc.

Transgenes are designated by a general formula:

TgX(YYYYY)#####Zzz where Tg indicates a transgene, and Xindicates the mode of insertion of the foreign DNA, with Nfor nonhomologous recombination, R for insertion via a viral vector, and H for homologous recombination. The YYYYY is a brief designation of the insert, with a range of standard abbreviations being available. The ##### is a laboratory assignment number. Finally, Zzz is a laboratory registration code. As an example, given in the rules:

C57BL/6J-TgN(CD8GEN)23Jwg

This is the designation for inbred strain C57BL/6J carrying a transgene with the human CD8 genomic (GEN) clone. It is derived from the 23rd mouse screened in a series of microinjection (N) in the laboratory of Jon W. Gordon (Jwg).

Breeding and Maintenance of Mutant and Transgenic Strains

The breeding methods needed to maintain these strains will depend on the mode of inheritance of the mutant, including whether it is dominant, codominant, or recessive; whether it has a distinct phenotype; and whether all classes of stock are viable and fertile. The genetic background (inbred or outbred) also needs to be taken into account. A brief description of some of the more common situations is given below. However, more complex breeding systems may be needed to produce animals with a desired combination of mutant genes, or where identification of genotype is difficult. Also, genetic mapping studies may involve quite complex breeding schemes not discussed here.

Both Sexes Fully Viable and Fertile — Such a mutant can be maintained according to the genetic background. If this is inbred, then methods for maintaining inbred strains, described above, should be used. If the mutant or transgene is on a heterogeneous, outbred background and there are no plans to backcross to an inbred strain, and if the aim is to maintain the colony for some time, then it should be maintained as for an outbred stock. In order to prevent inbreeding and genetic drift, at least 25 breeding pairs should be used with random mating, or about 13 pairs if a maximum avoidance of inbreeding system is to be used. Smaller colonies can be maintained for short periods, although it may result in substantial genetic drift and change in expression of the mutation.

A Recessive Mutation with One Sex Infertile — In this case, the mutant is usually maintained by mating a homozygous animal of the viable sex with a heterozygous animal of the other sex. This is the common situation with nude mice, where the females often have poor breeding performance (depending on genetic background), but males are fully fertile. Half the offspring will be of the desired mutant phenotype, and the other half will carry the gene and the appropriate sex can be used for further breeding. The matings need to take account of the genetic background that will be inbred or outbred, and should be maintained as such.

A Dominant Mutation with Homozygous Lethality — Some dominant genes such as the yellow allele at the agouti locus are lethal in the homozygous state. In this case, matings are made between mutant animals and wild type ones, with approximately half of the offspring being of the mutant phenotype. Again, account needs to be taken of the genetic background.

Recessive Mutation with Both Sexes Infertile — This is quite common with mutations which have serious phenotypic effects, such as the obese and diabetic mutations in mice. The problem is that if two heterozygotes are mated, on average, 1/4 of the offspring will be mutant, 1/2 will be heterozygous carriers, and 1/4 will be homozygous wild type. Unfortunately, the latter two classes will be phenotypically indistinguishable, but only the heterozygotes will be useful for further breeding.

The classical way of dealing with this situation is to set up test matings in order to identify heterozygotes. Known heterozygous animals can be crossed with individuals of unknown genotype. If mutant progeny are produced among a reasonably large number of offspring, then the animal of unknown genotype must be heterozygous. Unfortunately, this is time consuming and inefficient. Another alternative is to do random matings among the offspring of heterozygous matings. As 2/3 of the animals are, on average, heterozygous, 2/3 x 2/3 = 4/9 of the matings will be between heterozygotes, but only 1/4 of the offspring of such matings will be homozygotes. Generally, about 10 wild-type offspring will need to be produced to be reasonably confident that any particular mating is not between two heterozygotes, so can be discarded. Again, this is inefficient, though relatively simple.

If the mutant is on an inbred genetic background, an alternative method is to graft ovaries from a homozygous mutant animal into a wild-type female of the same inbred strain. This animal is then mated to a wild-type male to produce offspring known to be heterozygous for the mutation and suitable for breeding.

When a mutant locus has been sequenced and the nature of the mutation identified, it is often possible to develop a PCR-based method of genotyping individuals. All that is needed is a small sample of DNA and the laboratory capability of running the appropriate tests. Such a method has already been developed for the diabetes63 and obese mutations64 in mice, and is, or will be, available for many mutants. If heterozygotes can be identified in this way, then breeding mutant animals becomes much more efficient.

Breeding Transgenes and Mutations Produced by Gene Targeting ("Knockouts")

Carriers of a transgene can be of three types: hemizygotes, with one copy of the transgene but no normal host allele; heterozygotes, in the case of a gene-targeted mutation, with one mutant and one normal wild-type allele; or homozygotes, with two copies of the transgene. Identification of carriers usually presents no problems as there will be a PCR-based test or a probe that can be used to identify them using Southern blots. Thus, all that is needed is a sample of DNA. This is often obtained from the tail tip, though if a PCR method is used, an ear punch, hair sample, saliva,65 or even a fecal sample may provide sufficient DNA for the test. Once carriers have been identified, matings can be made to continue to maintain the transgene or backcross it to an inbred background or produce homozygous animals if the transgene is in a hemizygous or heterozygous state.

Problems may arise in differentiating between homozygous animals, i.e., with two copies of the transgene, and hemizygous or heterozygous animals, i.e., with one copy of the transgene. This will require a quantitative PCR66 or Southern blot analysis or a progeny test. The latter involves mating the animal to some wild-type animals and testing the progeny for the presence of the transgene, assuming the animal of interest is fertile and viable. If all the progeny carry a copy of the transgene (and assuming about eight to ten progeny have been tested), then the animal of interest can be assumed to have been homozygous.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment