This article is from the WSSF 2013 AFRMA Rat & Mouse Tales news-magazine.

Vasko Arosemena, e-mail
QI got to the AFRMA website thru a Google search, and I was simply fascinated with the stuff I found there. I’m doing a little research in the area of mouse genetics, but the majority of the information I’ve found on the Internet is really technical and difficult to absorb, especially for someone like me who is not an expert in genetics.

I basically wanted to know if it was possible to artificially create mice, either by microinjection of DNA or any other transgenic technology, that resemble other species color patterns, such as those found in cats and horses. If the answer is yes, could you lead me to Internet resources where I could find more information on the subject?

Answer by Carmen Jane Booth, D.V.M., Ph.D.
The ability to introduce foreign DNA (genes) from one species (i.e. humans or other species) into another species to produce genetically modified animals such as mice (transgenic mice) is common place in science and research today. For a basic understanding of this technology, the internet is rich with web sites explaining how this is accomplished. One that has good pictures is https://www.biology-pages.info/T/TransgenicAnimals.html.

So in answer to: Would it be possible to introduce a gene responsible for coat color pattern from domestic animals (dogs, cats, horses, etc.)? The answer is yes, the science and technology is possible.

We already see coat colors and patterns in mice that exist in other domestic species and humans. Albinism is caused by a variety of different mutations in one of the genes for enzymes (tyrosinase) in the pathway for conversion of tyrosine (not a pigment) to melanin (pigment).

For mice and rats with the Siamese coat color pattern seen in cats, this is a form of partial albinism. The mutated enzyme is heat-sensitive and fails to work at normal body temperatures (white coat color on body) and becomes active at the cooler areas of the skin on the head and extremities. Siamese cats who live in warmer climates have lighter coats than those who live in cooler climates. Wikipedia.org is a good source for information on coat color patterns in cats.

We see the same rich diversity of coat colors in mice and rats. Coat color patterns are caused by modifier genes. Because the question was specific to horse coat colors, I will digress and review coat color genetics in horses.

The following is adapted from: Coat color in Horses:
http://www.vgl.ucdavis.edu/services/coatcolorhorse.php

The basic coat colors of chestnut, bay, brown, and black horses are controlled by the interaction between two genes: Extension (gene symbol E) and Agouti (gene symbol A). The Extension gene (red factor) controls the production of red and black pigment. Agouti controls the distribution of black pigment either to a points pattern (mane, tail, lower legs, ear rims) or uniformly over the body. The effects of approximately 10 other genes may modify these pigments to provide an array of colors in the domestic horse ranging from white to black.

The basic colors can be diluted by at least five genes: Cream, Champagne, Dun, Pearl, and Silver. The Cream gene has a dosage effect in that a single copy of Cream produces palominos, buckskins, and smoky blacks. Two doses of Cream produce cremellos, perlinos, and smoky creams. Pearl is recessive; two copies of the gene or one copy of Pearl and one of Cream, are needed to see the dilution effect on the coat color. Champagne, Dun, and Silver do not show a dosage effect.

There are several genes responsible for white patterns on horses. White spotting patterns on the base coat color are produced by the Dominant White, Appaloosa, Tobiano, and Overo genes or as mixed white and colored hair patterns produced by the Grey (progressive whitening with age) and Roan genes. Several genes are involved in the production of white spotting patterns known as overo. Among those, the gene responsible for the frame-overo pattern is associated with a lethal disease of newborn foals called Lethal White Overo foal syndrome.

Detailed Coat Color Information

The Extension gene (red factor) has two alternative states (alleles). The dominant allele E produces black pigment in the coat. The recessive allele e produces red pigment. Red horses (chestnuts, sorrels, palominos, and red duns, to name a few) are homozygous, that is they have two alleles for the recessive red allele ee. Black pigmented horses (black, bay, brown, buckskin, and grullo, to name a few) have at least one E allele. They can be homozygous EE or heterozygous Ee. A horse that is homozygous EE will not produce red offspring, regardless of the color of the mate.

e	Only the red factor detected. The horse can be assumed to be homozygous for red (ee). The basic color is sorrel or chestnut, but depending on genes at other color loci, the horse could be palomino, red dun, gray, cremello, white, or any of these colors with the white hair patterns tobiano, overo, roan, or appaloosa.
E/e	Both black and red factors detected. The horse can be assumed to be heterozygous for the red factor (Ee). It can transmit either E or e to its offspring. The basic color of the horse will be black, bay, or brown, but depending on genes at other color loci, the horse may be buckskin, zebra dun, grullo, perlino, gray, white, or any of these colors with the white hair patterns tobiano, overo, roan, or appaloosa.
E	No red factor detected. The horse can be assumed to be homozygous for black pigment (EE). It cannot have red foals, regardless of the color of the mate. The basic color of the horse will be black, bay, or brown, but depending on genes at other color loci, the horse may be buckskin, zebra dun, grullo, perlino, gray, white, or any of these colors with the white hair patterns tobiano, overo, roan, or appaloosa.

The Agouti gene controls the distribution of black pigment. The dominant allele A restricts black pigment to the points of the horse (mane, tail, lower legs, and ear rims), as seen, for example, in bays and buckskins. The recessive allele a uniformly distributes black pigment over the entire body.

A/A or A/a	Black pigment distributed in point pattern. The basic color of the horse will be bay or brown in the absence of other modifying genes. A has no effect on red pigment (ee).
a	Only recessive allele detected. Black pigment distributed uniformly. The basic color of the horse will be black in the absence of other modifying genes.

Champagne is a dominant gene that dilutes hair pigment from black to brown and red to gold. Champagne on a chestnut background (Gold) produces a gold body color and often a flaxen mane and tail that can be mistaken for palomino. Champagne on a bay background (Amber) produces a tan body color with brown points. Champagne on a black background (Classic) produces a darker tan body with brown points. The skin of Champagne-diluted horses is pinkish/lavender toned and becomes speckled with age; the speckling is particularly noticeable around the eye, muzzle, under the tail, udder, and sheath. The eye color is blue-green at birth and darkens to amber as the horse ages. Champagne is inherited independently of other coat color genes and thus this dilution can occur in combination with any of the other genes that modify the base colors. Champagne dilution is found in Tennessee Walking Horses, Missouri Fox Trotters, Quarter Horses and related breeds, Miniature Horses, and Spanish Mustangs, among others. The increasing popularity of this color is making it more common in these breeds. A mutation in the Solute Carrier 36 family A1 (SLC36A1) gene was found to be associated with the Champagne dilution.

N/N	No evidence of the altered sequence detected
N/Ch	One copy of the altered sequence detected. Chestnut color (red) is diluted to gold, bay to tan with brown points, and black to darker tan with brown points.
Ch/Ch	Two copies of the altered sequence detected. All offspring are expected to be Champagne dilute.

Dun Zygosity Information

The Gray gene causes progressive depigmentation of the hair, often resulting in a coat color that is almost completely white by the age of 6–8 years. Horses that inherit progressive Gray can be born any color, then begin gradually to show white hairs mixed with the colored throughout the body. Usually the first signs of gray hair can be found on the head, particularly around the eyes. Gray is dominant, therefore a single copy of this gene will cause a horse to turn gray. If a horse has two copies of Gray, all offspring of this horse will be gray. Research indicates that horses with one copy of Gray often retain some of the original pigment while homozygotes tend to progress to almost completely white. Gray is found in many breeds and is the predominant color of the Lipizzaner breed.

N/N	No copies of the gray gene. Horse will not turn gray.
N/G	One copy of the gray gene. Horse will turn gray and approximately 50% of offspring will be gray.
G/G	Two copies of the gray gene. Horse will turn gray and all offspring will be gray.

Horses have four common coat color dilution genes with defined phenotypes: Cream, Dun, Silver, and Champagne. Two rare dilution phenotypes have been recognized in Quarter Horses and Spanish horse breeds such as Andalusians and Lusitanos. In Spanish horses, this dilution is known as Pearl. In Quarter Horses and Paints, it has been commonly known as Barlink Factor and the genetic mutation is known. The presence of this mutation in Quarter Horses and Paints likely reflects the Spanish horse ancestry of these modern breeds. To recognize that this mutation probably originated in Spanish horses, it is appropriate to name it Pearl.

Pearl behaves as a recessive gene with respect to the hair color. One dose of the mutation does not change the coat color of black, bay, or chestnut horses. Two doses on a chestnut background produce a pale, uniform apricot color of body hair, mane, and tail. Skin coloration is also pale. Pearl is known to interact with Cream dilution to produce pseudo-double Cream dilute phenotypes including pale skin and blue/green eyes.

N/N	No evidence of altered sequence detected.
N/Prl	One copy of the altered sequence detected. If Cream dilution is also present, a pseudo-double Cream phenotype will result.
Prl/Prl	Two copies of the altered sequence detected. On a chestnut base color, a uniform apricot color of body hair, mane, and tail will result.

The horse Silver dilution gene dilutes black pigment but has no effect on red pigment. The mane and tail are lightened to flaxen or silver gray, and may darken on some horses as they age. A solid black horse with this gene will be chocolate colored with a lightened mane and tail. A bay horse will have the black pigment on the lower legs, mane, and tail lightened. Sometimes bay horses with Silver dilution can be mistaken for chestnuts with a flaxen mane and tail. Silver dilution is inherited as a dominant trait. It is known to occur in Rocky Mountain horses and related breeds, Shetland ponies, Icelandic, and Morgan horses.

The gene responsible for Silver dilution has been recently identified as PMEL17 by researchers in Sweden. Two single nucleotide substitutions have been found to be associated with the dilution, one in intron 9—A (normal) to T (silver)—and the other in exon 11—C (normal) to T (silver). VGL’s test for Silver dilution assays both sites.

N/N	No evidence of altered sequence detected.
N/Z	One copy of the altered sequence detected. Black- based horses will be chocolate with flaxen mane and tail. Bay-based horses will have pigment on lower legs lightened and flaxen mane and tail. No effect on chestnut color.
Z/Z	Two copies of altered sequence detected. Black- based horses will be chocolate with flaxen mane and tail. Bay-based horses will have pigment on lower legs lightened and flaxen mane and tail. No effect on chestnut color.

In horses, the KIT gene has crucial function for the development of blood, gonadal, and pigmentary tissues. Mutations that affect normal functioning of KIT gene products often result in lack of pigment cells (melanocytes) in the skin and hair follicles which leads to white patterning in horses known as dominant white or white. Dominant white expression ranges from a minimal Sabino-like pattern to a completely white phenotype. Studies of inherited white phenotypes in different breeds of horses have shown that these arise as independent mutations. To date, 14 different KIT mutations have been identified that are associated with white patterns. In 2000, a Quarter Horse named GQ Santana was born with a KIT gene white mutation (called W10) and became a prominent stallion in both AQHA and APHA. Horses with the W10 mutation have pink skin where the hair is white and the eyes have normal pigmentation and are rarely blue. Because of the nature of the molecular change, it is thought that only horses that carry one copy of W10 are viable, but this remains to be confirmed.

N/N	No evidence of altered sequence detected.
N/W10	One copy of the W10 mutation detected. Horse will display some degree of white spotting but the specific pattern cannot be predicted.
W10/W10	Two copies of the W10 gene detected—to date, no viable offspring have been produced.

Horse breeding programs specializing in overo have particular challenges compared with programs for other white patterns such as tobiano. Not only is there the possibility of producing a solid dark foal without the overo pattern but there is also the risk of producing an all-white foal that dies of complications from intestinal tract abnormalities (ileocolonic aganglionosis). As far as we are aware, overo horses themselves have no specific health risks. While breeding evidence shows that some overos are heterozygous for a gene that is lethal in the homozygous condition, it has not been easy to identify which horses have the overo gene that is associated with the lethal white foal syndrome. Occasionally, even solid-colored horses without obvious body spotting patterns have been reported to produce lethal white foals. Clearly the spotting pattern classified as overo is phenotypically and genetically heterogeneous [different].

Using the letter O to symbolize the DNA sequence of the lethal white (LW) overo gene and N for the sequence of the non-overo, then the lethal white foals can be symbolized as OO, their overo parents as NO, and non-overos as NN.

Breeding predictions between LW overos (NO x NO):

	N	O
N	25% NN solid	25% NO overo
O	25% NO overo	25% OO lethal

Breeding predictions between LW overo and solid (NO x NN): No possibility of lethal white foals.

	N	O
N	50% NN solid	50% NO overo

Sabino is a generic description for a group of similar white spotting patterns. The sabino pattern is described as irregular spotting usually on the legs, belly, and face, often with extensive roaning. A mutation has recently been discovered that produces one type of sabino pattern. It has been named Sabino1 as it is not present in all sabino-patterned horses. More mutations will probably be identified that account for other sabino patterns.

Sabino1 is inherited as an autosomal dominant mutation. One copy of the Sabino1 gene is expected to produce horses with two or more white legs or feet—often with white running up the anterior part of the leg, an extensive blaze, spotting on the midsection, with jagged or roaned margins to the pattern. Horses with two copies of the Sabino1 gene are at least 90% white and are referred to as Sabino-white.

Sabino1 is most commonly found in Tennessee Walking Horses. Other breeds in which this mutation has been found include: American Miniature Horses, American Paint Horses, Aztecas, Missouri Fox Trotters, Shetland Ponies, Spanish Mustangs, and Pony of the Americas. Other breeds of horses that are known to have sabino patterns, such as Clydesdales and Arabians, have so far tested negative for the Sabino1 mutation, although the number of animals tested is low.

N/N	No evidence of altered sequence detected.
N/SB1	One copy of the Sabino1 gene detected. Horse typically may have two or more white legs, blaze, spots or roaning in the midsection and jagged margins around white areas.
SB1/SB1	Two copies of the Sabino1 gene detected. Complete or nearly complete white phenotype expected.

Splashed white is a variable white spotting pattern characterized primarily by an extremely large blaze, extended white markings on the legs, variable white spotting on the belly, and often blue eyes. Some, but not all, splashed white horses are also deaf. Recent research has identified three mutations—SW-1, SW-2, and SW-3—that cause splashed white phenotypes in horses. SW-1 has been found in several breeds. Horses homozygous for SW-1 (SW1/SW1) have been identified, which suggests that this mutation is not homozygous lethal. SW-2 and the rare SW-3 occur exclusively in certain lines of Quarter Horses and Paints. Based on predictions from other species, SW-2 and SW-3 may be homozygous lethal and thus matings of two horses that carry SW-2 or SW-3 should be avoided.

Horses that carry combinations of the splashed white mutations, tobiano, or lethal white overo can display extensive white patterning or be white.

N/N	No copies of SW-1 mutation
N/SW1	Horse has one copy of the SW-1 mutation
SW1/SW1	Horse has two copies of the SW-1 mutation
N/N	No copies of SW-2 mutation
N/SW2	Horse has one copy of the SW-2 mutation
N/N	No copies of SW-3 mutation
N/SW3	Horse has one copy of the SW-3 mutation

Research about the splashed white is still in progress.

The tobiano white spotting pattern is a trait controlled by a dominant gene. The pattern is clearly marked and characterized by white across the spine that extends downward between the ears and tail. The skin underlying the white spots is pink and under the colored areas it is black. The eyes are usually brown, but one or both may be blue or partially blue. The head is dark, with white markings like those of a solid colored horse. Usually, all four legs are white below the hocks and knees. The spots are generally regular and distinct as ovals or round patterns. The tail can be two colors—a characteristic seldom seen in horses that are not tobiano. A tobiano can be predominantly dark or white.

The tobiano gene has two alternative states (alleles). The dominant allele, TO, produces the tobiano pattern and the recessive allele, to, is non-tobiano (called N). A horse that is homozygous for tobiano, symbolized as TO/TO, will always produce offspring that are tobiano regardless of the mate.

Unlike other white spotting patterns caused by specific changes in DNA sequence of the genes, Tobiano is associated with a large chromosome inversion that affects the function of the gene KIT. The inversion associated with the Tobiano pattern was identified by researchers at the University of Kentucky.

N/N	No evidence of altered sequence detected. Horse is not Tobiano
N/TO	One copy of altered sequence. Approximately 50% of the offspring will inherit Tobiano
TO/TO	Two copies of altered sequence. Horse is homozygous for Tobiano. All offspring will inherit Tobiano

Most color assignments can be correctly made based on physical appearance or phenotype alone. However, genetic testing may be necessary to define phenotypes that are visually ambiguous or the color possibilities for offspring. Researchers at the Veterinary Genetics Laboratory and other institutions are working towards the identification of the specific genes and mutations responsible for coat color traits in the horse.

The genetic defect responsible for the Lethal White Overo in horses occurs in mice. This gene causes failure of migration of neural crest cells during embryogenesis. The coat color pattern is called piebald and the mice were referred to as lethal spotted white mice (Migration of ganglion cell precursors in the ileoceca of normal and lethal spotted embryos, a murine model for Hirschsprung disease. Coventry S, Yost C, Palmiter RD, Kapur RP. www.ncbi.nlm.nih.gov/pubmed/8041122). These neural crest cells ultimately migrate to form the neurons in the intestine. Failure of migration of enteric ganglion cells in the intestine causes the disease known as Hirschsprung disease. I worked with these mice while an undergraduate at U.C. Davis before the genetics were known. Unfortunately, the gene responsible for it can be lethal if homozygous. There are mutations in at least 13 different genes associated with Hirschsprung’s disease in humans. In mice, as in horses, the genetic mutations are also associated with white coat color. Mice genetically black and with the mutation have varying degrees of white spots (100% white is lethal in mice [and horses]) because the mutations are recessive. To be 100% white, the mouse or foal would need two copies of the recessive gene. By coincidence, during graduate school I rotated in the laboratory of the investigator who ultimately determined the gene that was responsible for this in mice (Transgenic Rescue of Aganglionosis and Piebaldism in Lethal Spotted Mice, Julie Rice, et al., Developmental Dynamics 217:120–132, 2000).

Coat Color in Mice*

Coat color genetics is well-characterized for mice. Although there are a great number of loci which influence the synthesis of melanin in mice, there are only two loci which control the nature of the pigment formed. Thus the agouti and extension series of alleles determine the relative amount and distribution of yellow pigment (phaeomelanin) and black or brown pigment (eumelanin) in the hairs of the coat. The agouti locus (chromosome 2) is particularly interesting because it is a coat-color determinant which acts via the hair follicle. It also appears to be a complex locus. The number of alleles which have been described as being part of this complex—17 in all—and the number of reported mutations, also exceeds that recorded for any other locus concerned with the synthesis of melanin. For a better understanding of coat color in mice, please see The Jackson Laboratory web site.*

So, before getting back to the question, one must remember that different species have a different number of diploid chromosomes—humans have 46, horses have 64, and mice have 40. The chromosomal location of specific genes varies by species. Because the entire genome for mice, dogs, and horses is complete, it would be possible to find out if mice have the same genes as horses, and one could make genetically altered mice with the gene or the specific mutations in horses or dogs that have a coat color phenotype. The cost to make a transgenic mouse varies from $4,500 and up by institution.

Finally there is one more issue to consider and that is the structure of the hair in mice and horses—they are in fact very different. Horses have single hair follicles that produce only one hair at a time. In contrast, dogs and cats have compound hair follies that produce both primary and secondary hairs. One has only to think of northern breeds of dogs who shed their undercoat.

Mouse Coat Development*

Any consideration of coat color requires some appreciation of the intimate relationship that exists between the melanocyte and the hair in which its product, the melanin granule (or mature melanosome), is deposited. Therefore, before embarking on the main theme of this effort, it is necessary to comment briefly on the coat of the mouse and its development.

Mice on the other hand, while they do have compound hair follicles, they also have two kinds of hair—overhairs and under hairs. They have three types of overhairs (which together make up about 20% of the total number of hairs)—the guard hairs (or monotrichs), the awls, which have no constrictions, and the auchenes, which have a single constriction. The underhairs or zigzags constitute the remaining and predominant hair population. These fibers are shorter than the other hair types and usually have three flat constrictions, the segments following each being angulated against each other (and hence zigzagged). Because of their small size, these underhairs play only a minor role in determining the overall color of the animal.

In fine structure, all hairs are essentially similar, consisting of a wide central medulla surrounded by a narrow cortex which, in turn, is surrounded by a thin cuticle. The tips and bases of all the hairs are solid and deficient in medullary cells, and these cells may also be absent at the constrictions. Other than in these regions, the medullary cells are arranged transversely, separated from one another by areas devoid of melanin granules. In the overhairs these medullary cells may form rows of three, four, or even five septules, whereas in zigzags there is only a single row of septa. Although pigment granules are normally present both in the cortex and in the medulla of the shaft, most of the pigment occurs in the medulla. The four main hair types and some of their characteristics are shown in Figure 1.

Figure 1. (a) Diagram of a longitudinal section of a hair. (b) The four main hair types drawn to scale (approx., x9). Arrows indicate where photographs were taken for (c) (approx., x400). (b) and (c) from H.B. Chase and H. Rauch, 1950. Reproduced with permission of the authors and The Wistar Institute Press.

Conclusion: So while it is possible to put genes from horses that affect coat color or patterns into mice, it is unknown if the anatomical and developmental differences in the actual hairs would be able to be replicated in mice from what is seen horses (Figure 2.).

Figure 2. Hair Shafts: Left Mouse and Right Horse Used with permission from http://micro.magnet.fsu.edu.

*Adapted from The Jackson Laboratory
http://www.informatics.jax.org/wksilvers/frames/frame1-1.shtml

Ed. Note: Some additional reading:

Mosaic expression of a tyrosinase fusion gene in albino mice yields a heritable striped coat color pattern in transgenic homozygotes, Beatrice Mintz and Monika Bradl, Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 9643–9647, November 1991, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC52774/pdf/pnas01071-0277.pdf

Clonal coat color variation due to a transforming gene expressed in melanocytes of transgenic mice, Monika Bradl, Lionel Larue, and Beatrice Mintz, Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 6447–6451, August 1991, www.ncbi.nlm.nih.gov/pmc/articles/PMC52102/pdf/pnas01065-0069.pdf.

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