2009 Group Project 1
- 1 THE RABBIT (ORYCTOLAGUS CUNICULUS)
- 2 Introduction
- 3 History of Model Use
- 4 Timeline of Embryo Development
- 5 Staging
- 6 Genetics
- 7 Abnormal Development
- 8 Current Embryology Research
- 9 References
- 10 Glossary
- 11 Links to Research Laboratories and Researchers
- 12 ANAT2341 group projects
THE RABBIT (ORYCTOLAGUS CUNICULUS)
Several characteristics of the rabbit make it an excellent model for study. This will be further explored in "The History of Model Use" section of this page. Many studies have resulted in the development and improvement of various micro-manipulation techniques such as the production of transgenic rabbits. Unlike many other species such as the chick or rat, relatively little is known about the development of a rabbit. Nevertheless, it is still an appropriate animal model as the results from many experiments are significant to that of other mammals, including humans. (55)
A rabbits potential for reproduction is high, breeding from the early stages of 3 to 4 months of age. A mature female rabbit can be pregnant from 6 to 8 months in a year, producing up to 30 to 40 young in this time. (56)
History of Model Use
WHY are we using rabbits?
- Provides repeatability of animal model studies
- Large enough for single samples
- Many stocks/strains as animal models
- Easily managed
- Quality of immunologic products
- Ease of reproductive control
- Most colonies are a storehouse of diseases
- Extremely variable to responses to general anesthetics
Brief timeline of rabbit embryo model use
- 1672- de Graaf found the Graffian follicle.
- 1890- Walter Heape succeeded first mammalian embryo transfer
- 1906- FT Lewis discovered the development of the lymphatic system in rabbit embryos.
- 1941- Dr. Pincus succeeded in keeping rabbit embryos developing in the test
- 1968- Edwards and Gardner successfully performed the first known embryo biopsy on rabbit embryos
Regnier de Graaf (1641–1673)
Discovery of the Graafian Follicles
In 1672 de Graaf published The Generative Organs of Women, which was primarily a study of development in the rabbit. When de Graaf discovered large, round welling on the ovaries of rabbits, he assumed they were mammalian eggs. De Graaf also described the corpus luteum.
He summarised the previous works from anatomists, but unable to experience the amazing benefits made by microscopy. But Antonie van Leeuwenhoek (A microbiologist, 1632 - 1723) argued that the structures now known as Graafian follicles could not be eggs. Haller suggested that the egg might be formed by the coagulation of the fluid within the Graafian follicle. De Graaf noted that the “egg” did not contain a tiny embryo, but he thought it did contain the “germ” of the future organism. (3)
Walter Heape (1855-1928)
First case of embryo transfer experiments
On 27 April 1890, Walter Heape (a professor and physician at the University of Cambridge, England) transferred rabbit embryos from one mother to another. (7) One rabbit mother became pregnant and delivered young from the transferred embryos. This was the first mammalian embryo transfer experiment to be successfully completed. His embryo transfer work in perspective as it relates to other contributions of this pioneer in reproductive biology.(7)
In 1891, Walter Heape had been conducting research on reproduction in numerous animal species. Working with two species of rabbits, he flushed embryos from the rabbit fallopian tubes of one breed (Angora) and placed them into the uterus of a recently mated Belgian hare. In the resulting litter, there were 4 Belgians and 2 Angoras. Heape proved that it was possible to take preimplantation embryos and transfer them to a gestational carrier without affecting their development.(7)
Dr. Gregory Goodwin Pincus (1903-1967)
Dr. Pincus began studying hormonal biology and steroidal hormones early in his career. His first breakthrough came when he was able to produce in vitro fertilization in rabbits (by using chemicals) in 1934. Throughout their hormonal contraceptive research Pincus, along with reproductive physiologist Min Chueh Chang, found out progesterone would act as an inhibitor to ovulation. They co-invented the combined oral contraceptive pill.
In 1968, Robert Edwards and David Gardner reported the successful sexing of rabbit blastocysts, setting the first steps towards PGD (Preimplantation Genetic Diagnosis). It was not until the 1980s that human IVF was fully developed, which coincided with the breakthrough of the highly sensitive polymerase chain reaction (PCR) technology. Handyside and collaborators' first successful attempts at testing were in October 1989 with the first births in 1990 though the preliminary experiments had been published some years earlier. In these first cases, PCR was used for sex determination for patients carrying X-linked diseases.(12)
Timeline of Embryo Development
The following is a timeline, adapted from Cibelli (2002), of the main events of preimplantation development in rabbits. The time is measured in hours post-mating and shows the embryo stage (cell number) in each time scale.
12-14 hours: Oocyte; Fertilization
18-20 hours: Zygote; Pronuclear formation
24-26 hours: Two cell
30-32 hours: Four cell
38-40 hours: Eight cell; Maternal-zygotic transition
46-48 hours: Sixteen cell
54-56 hours: Morula (32 cell); Compaction and transport to uterus
64-66 hours: Compact morula (64 cell); Morula-blastocyst transition
76-78 hours: Early blastocyst (128 cell)
84-86 hours: Expanded blastocyst (256 cell); Blastocoels expansion
94-96 hours: Hatched blastocyst (512 cell); Hatching
The following shows a diagrammatic representation of the relative sizes of rabbit embryos. The sizes do not include the embryonic coverings such as the zona pellucida and mucin coat. This diagram has been adapted from Warner (2003).
The following timelines show a comparison of the developmental stages between human and rabbit embryos. They have been adapted from Derelanko (2008). "The similarities of this developmental pattern in humans and rabbits, suggests that the same growth increment is required to achieve the same stage. The main difference observed between human rabbit gestational duration is due to the fetal growth phase. The reason for this may be due to birth weight, lifetime and the neural complexity of the species." (59)
The following timelines have been adapted from a study carried out by Beaudoin et al. on the development of rabbit embryos.
8.5 days: Embryo thickens. First somites appear. Rostral neuropore closes. Caudal neuropore remains open
9.5 days: Dorsal curvature begins. Cardiac mass bulges under cephalic pole.
10.5 - 13.5 days: Dorsal curvature increases. Body thickens. Softening of the cephalic domination straightens the embryo.
17.5 days: Neck becomes visible.
9.5 days: Rostral limb bud appears
10.5 days: Caudal limb bud appears
12.5 days: Hand plate becomes present. Limbs become disposed.
13.5 days: Foot plate and finger rays become visible.
14.5 days: Rostral and caudal limbs become parallel.
15.5 days: Elbow appears.
16.5 days: Fingers start to elongate.
17.5 days: Knee becomes visible
19.5 days: Hands and feet merge on the midline. Three segments of limbs become distinguished.
Abdominal Wall Development
9.5 days: Abdominal wall is limited to the embryonic pedicle under the cardiac mass.
13.5 days: First intestinal loops appear in the umbilical cord.
14.5 days – 17.5: Rapid intestinal development in the cord.
15.5 days: Abdominal vesicles can be observed.
16.5 days: Cecal bud becomes present outside the abdomen
18.5 days: Bowel returns to the abdominal cavity and umbilical ring closes.
9.5 days: One cerebral vesicle can be seen. Pharyngeal arches as optic vesicle are present.
10.5 days: Three arches are distinguished in the cephalic pole.
11.5 days: Three cerebral vesicles present. Optic plate is distinguished.
12.5 days: Face develops nasal, maxillar and mandibular buds. Lens vesicle is closed. Five cerebral vesicles exist.
14.5 days: The ear becomes refined. Face can be distinguished from the brow.
18.5 days: Eyelid appears, covering the eyes.
The following stages have been adapted from Beaudoin et al. (2003). Table 1 represents at each age its corresponding stage defined by the Carnegie classification
The National Human Genome Research Institute selected the European rabbit (Oryctolagus cuniculus) for whole genome sequencing to enhance their understanding of the human genome and use it experimentally for an animal model for human disease. (8)
The rabbit has been sequenced twice by The Broad Institute as part of the mammalian genome project. It is now currently undergoing 7 more sequencing projects. Its sequencing is made by the Whole Genome Shotgun (WGS) and assembly method. (9) This is when genomic DNA is sheared into small pieces of approximately 2000 base pairs which are then cloned into plasmids and sequenced on both strands. Once the contig fragments are read, realigned and reassembled by computer algorithms, it will give the overall sequence. (10) (11) The image below shows diagramatically how the two types of sequencing are different. The same techniques are used for sequencing the human genome (in 2003). [A contig is a set of overlapping DNA segments, derived from a single source of genetic material, from which the complete sequence may be deduced.
The whole genome shotgun (WGS) has serious gaps, yet the information has already proven useful for immunological as well as in silico studies. Deeper 7x coverage started in September 2007. The NCBI Rabbit Genome Resources site has links to searches for genes in the assemblies of the 2x WGS sequence at Ensembl and UCSC.(12) Rabbit Genome Project
The Rabbit genome was published by two groups (9)
- Lindblad-Toh,K., Chang,J.L., Gnerre,S., Clamp,M. and Lander,E.S. published their admission of 84024 bases on May 5th 2005 to The Broad Institute (USA) by shotgun sequencing
- Di Palma,F., Heiman,D., Young,S., Gnerre,S., Johnson,J., Lander,E.S. and Lindblad-Toh,K. published their admission of 84024 bases on August 3rd 2009 to The Broad Institute (USA) by shotgun sequencing.
The rabbit's genome is sequenced and on display in the Nucleotide Data Bank. It is too long to produce here. A link to the data bank is provided: Rabbit Genome from the Nucleotide Data Bank
The rabbit genome was sequenced in 2005 by Ensembl and managed to produce: (13)
- 2,076,044,328 supercontigs (ordered Contigs with gaps)
- 495 Known protein-coding genes
- 11,357 Projected protein-coding genes
- 2,343 RNA genes
- 212,581 Gene exons
- 20,311 Gene transcripts
For further research the taxonomy ID number for the Rabbit is: 9986 Taxonomy Data
The mitochondiral genome (mtDNA) of the rabbit was sequenced on November 14th 2006 with 17245 base pairs/nucleotides in circular form. Apparently the "length is not absolute due to the presence of different numbers of repeated motifs in the control region". PMID 9653643
The image to the right shows the mitochondria during cell division and multiplication in embryo development.
Over the years there have been various data suggesting the diploid chromosomal number for the rabbit may range from 22-42. However through recent research and an abundance of trials suggest that the rabbit does indeed have 22 different chromosome pairs existing in each cell of the rabbit. (16)(17) The sex chromosomes of the rabbit are of X-Y type convincingly (17). In a study in Bombay it confirmed that a rabbit has 2n = 44 chromosomes. There were 21 pairs of autosomes, out of which 1 to 6 were metacentric, 7 to 11 submetacentric, 12 to 17 subtelocentric and 18 to 21 acrocentric, plus the sex chromosomes. The image to the left shows a female rabbit with no chromosomal abnormalities. (19)
Comparison to human chromosome:
Rabbit chromosomes 12, 19 and X were found to be completely homologous to human chromosomes 6, 17 and X, respectively. All other human chromosomes were homologous to two or sometimes three rabbit chromosomes. (14) Chromosome 12 was shorter than chromosomes 13 and 14. (15) The image below illustrates the human chromosome (2n = 46).
|----| is approximately 2800nm
- |---| is approximately 1300nm
These are generally more apparent during early embryo development in blastocysts from delayed fertilization. Some examples of abnormal development include cases of: (18)
- hypoploidy; 1 chromosome missing from a pair (2n = 43),
- double hypoploidy; 2 chromosomes missing from 2 different pairs (2n = 42),
- mosaicism (different chromosomal makeup in some cells).
Another study also found these abnormalities as well as: (19)
- autosomal trisomy (3 autosomes per pair),
- triploidy (extra set of chromsomes),
- mixoploidy (unequal number of chromosome sets in adjacent cells), and
- short arm deletion (deletion of parts of chromosomes).
A specific abnormality occurring in rabbits is the x-linked tremor. There is a mutation in exon 2 of the prteolipid-protein (PLP 1) gene, corresponding to the end of the first potential transmembrane domain of the protein. This disorder affects myelination of the central nervous system. OMIA ID:12 Gene:100009169
Abnormal embryological development is a vast field of study that has been the subject of recent research papers. Our investigation of abnormal development in rabbit embryology will focus on abnormalities commonly found in both rabbit and human embryos. We will explore the nature of these abnormalities in both humans and rabbits.
Annually, one in every 1000 children born in Australia have hydrocephalus. Hydrocephalus occurs when excessive cerebrospinal fluid (CSF) accumulates in the brain and can result in severe disability and even death because the disorder can result in complete or near complete destruction of the cerebral cortex (22).
Children born with hydrocephalus typically exhibit abnormally large head circumference and bulging cranial fontanels as a result of increased intracranial pressure on the brain from the accumulating CSF (22,23). As CSF accumulates in the ventricles and CSF compartments of the brain, it expands forcing the brain to grow outward. The outward growing brain places pressure on the skull which in turn also grows outwards giving rise to an abnormally large head circumference and bulging cranial fontanels (22,24). It is generally acknowledged that children with hydrocephalus have mental retardation, often to the degree of being “vegetative” (24).
The serious consequences of this disease mean that much research is necessary to unearth methods of prevention and effective treatment. One of the modes of studying hydrocephalus is to use the rabbit experimental model where hydrocephalus can be induced in rabbit embryo’s. The injection of silicone oil into the cisterna magna of the brain is one way of inducing hydrocephalus in the rabbit embryo. The silicone oil obstructs the normal flow of CSF resulting in CSF accumulation in the brain leading to hydrocephalus (25). Another method of inducing hydrocephalus in rabbit embryo’s is by intentional vitamin A deprivation of pregnant dams. The vitamin A deficiency results in raised intracranial CSF pressure and aqueduct stenosis causing poor circulation of CSF leading to hydrocephalus (26,27). Although the exact function of vitamin A on brain development and CSF regulation is not fully understood, it is clear that Vitamin A has an important role in brain development and its normal functioning (27).
In Australia, the risk of spina bifida is 1 in every 500 pregnancies. Spina bifida is a type of neural tube defect where vertebrae (which normally cover and protect the spinal cord) are not completely formed but are divided resulting in the defective spinal cord and its coverings to protrude through the opening (28,29).
There are three main types of Spina Bifida differentiated by their characteristic features:
1)Spina Bifida Meningocele characterized by normal spinal cord, divided outer vertebrae and meninges surrounding the spinal cord protruding from the divided vertebrae as a cyst (1,28).
2)Spina Bifida Myelomeningocele characterized by split outer vertebrae with spinal cord and its meninges protruding from the divided vertebrae as a cyst. Commonly found at lumbar vertebral level (1,28,29).
3)Spina Bifida Occulta characterized unfused vertebral arches and exposed vertebral canal. Spinal cord and its meninges still located in vertebral canal (1,28).
All forms of spina bifida are potentially fatal and in those where the spinal cord is damaged severe consequence arise including and paralysis and loss of sensation at and below the level of damage spinal cord damage (29,33). There is a lot of Spina bifida research using rabbit models to test the effective methods of correcting spina bifida. The occurrence of natural spina bifida in rabbits is rare and for experimental purposes it can be surgically created in rabbit fetuses during gestation (31,32). The process involves aesthetical sedation of the maternal rabbit at day 22 of gestation; a midline laparotomy performed and the desired type of spina bifida lesion created on the exposed fetus using forceps. Different methods for correcting spina bifida can then be tested and its effectiveness analysed (30,33,34).
Brachydactylia & Acheiropodia
Brachydactylia and Acheiropodia are genetic disorders characterized by skeletal malformation of the hands and feet (36,36). In Brachydactylia, the malformations commonly involve abnormal shortening of fingers and toes due to poorly formed or absent bones (35). Acheiropodia is characterized by more severe skeletal malformations including bilateral amputations of the distal upper and lower extremities as well as aplasia of the hands and feet. Although it is not fatal, the individual endures a very difficult life without hands and feet (37).
The inheritance mechanism of this diseases can has been studied through experiment using rabbits. Rabbits with abnormal genes coding for Brachydactylia and Acheiropodia are selected and bred to produce offspring with the disease (36). Brachydactylia and Acheiropodia are autosomal recessive disorders which means that two copies of an abnormal gene must be present in the affected individual in order for the disease to develop. Thus, each parent passes an abnormal gene to the offspring (35,36). The process of the malformation progresses from genotype to phenotype; small deletions on the chromosomes produce abnormal genes, the abnormal genes are then passed down to the offspring, the offspring that inherits two of the abnormal genes is unable to code for the correct proteins and as a result, there is failure in normal development of limb extremities in the embryo phenotype (37,38).
Current Embryology Research
There is currently great excitement in research involving rabbits in the fields of transgenesis, cloning and stem cells. We shall explore research in these areas by discovering some of the techniques used and how they have been applied to the rabbit model.
A transgenic organism is one whose genome also contains genes from another species (39). The aim of creating a transgenic organism is to obtain a favorable characteristic in the organism’s phenotype (41). This desired characteristic of phenotype is obtained by altering the organism’s normal genotype to include the gene from another species with the desired characteristic (39,40).
The production of a transgenic organism involves several steps summarized below:
1) The chromosome and the desired gene on it are identified in an organism (39).
2) The gene is isolated from its DNA strand. This involves “cutting” it out of its DNA strand using enzymes called restriction endonucleases. The restriction endonucleases cut DNA at specific site so the desired gene can be removed from the DNA strand. The cut ends are known as “sticky ends” (39,40).
3) Separate DNA sequences for regulation sometimes have to be added to ensure the gene will work (40).
4) The gene is then inserted with a promoter sequence into the fertilized egg cell of a new organism producing recombinant DNA (39,44). To do this, firstly the new organism’s cell DNA must first be cut and the desired gene incorporated into it. The same enzymes are used to cut the new organism’s cell DNA because the DNA stands from the two different organisms will form matching sticky ends that will be attracted to and connect with each other in a process is called “annealing” (39,40,44).
5) DNA ligases are sealing enzymes found in all living organisms that help make and repair DNA (39,40). The DNA ligases are added to the annealed DNA fragments to help strengthen the bonds of the new recombinant DNA. The recombinant DNA is now transferred into the new organism’s fertilized egg by microinjection (44).
6) As the embryo develops and the recombinant genetic code read, new proteins are synthesized which code for the new desired phenotype characteristic in the transgenic organism (39,40).
Transgenic rabbits can be created in the laboratory by gene microinjection into the fertilized rabbit oocyte (44). In April 2000, a transgenic rabbit named Alba was born containing a gene from a Pacific Northwest jellyfish. The gene which was injected into a fertilized albino rabbit oocyte allows the rabbit to synthesize the green fluorescent protein (GFP) that is characteristic of the Pacific Northwest jellyfish. When illuminated correctly, Alba glows a bright green (43,46).
Transgenic rabbits have important research purposes. Transgenic rabbits can be created to model retinal degeneration (44). Retinal degeneration is a common problem with aging and diseases such as diabetes, and frequently leads to complete blindness (42). A genetic retinal degeneration disease called retinitis pigmentosa (RP) is created in these rabbits by introducing the “Pro347Leu” mutation of the rhodopsin gene into fertilized rabbit eggs (43,44). As the rabbit grows, it develops the disease which becomes progressively worse (45).The use of these transgenic rabbits ultimately leads to better understanding of the disease and more effective treatments (42).
Cloning is a method of producing genetically identical organisms. The principle of cloning relies on “tricking” an egg cell to begin rapidly dividing into an embryo (39). In the normal fertilization process, two haploid sex cells unite to form a diploid zygote which begins rapidly dividing into an embryo (40). By replacing the haploid egg cell nucleus with a diploid somatic cell nucleus, the egg cell is tricked into thinking it is fertilized and hence begins rapidly dividing to form an embryo (39,40,41).
The process of cloning involves several steps summarized as the following using a rabbit example:
1) A somatic cell is obtained from a rabbit and the nucleus removed (39).
2) An egg cell is obtained from a donor rabbit and the nucleus of the egg cell removed using an enucleation needle. The cell is now called an enucleated egg cell (egg cell without a nucleus) (39,41).
3) The nucleus of the somatic cell is inserted into the enucleated egg cell (41).
4) The cell is now stimulated to divide by applying pulses of electric current (39).
5) The rapidly dividing cell develops in culture for a few days forming the early embryo (40,41).
6) The embryo is then implanted into the uterus of another rabbit similar to the egg cell donor. This rabbit is called the surrogate mother (40,41).
7) The embryo develops and the surrogate mother gives birth to a rabbit fetus with near identical genotype of the somatic cell donor (the small genomic difference is caused by the fetus obtaining mitochondrial DNA from the egg cell donor) (39,41).
The cloning of rabbits has been achieved by inserting the diploid nucleus of a rabbit fibroblast (the principal cells in connective tissue) into an enucleated oocyte (49). There is currently much focus on the creation of controversial but revolutionary interspecies hybrid embryos where the nucleus from a human somatic cells are implanted into an enucleated egg cell of another animal (50). The resulting embryo (known as a chimera) will be almost completely human genotype but will have genome from the other animal (48). In 2003, scientists at the Shanghai Second Medical University fused human cell with rabbit eggs creating the first human-rabbit hybrid using the nucleus transfer technique. The hybrid embryo developed in culture for a few days before it was destroyed and the stem cells harvested (47,48). The purpose of the research into creating hybrid embryos is to provide researchers with human stem cells for experimentation into cures for diseases such as cystic fibrosis, Alzheimer’s and Motor Neuron disease as human stem cell are difficult to obtain (50).
Stem cells are unspecialized cells which are able to continuously reproduce themselves and under appropriate conditions, differentiate into all the various specialized cells of the organism such as cardiac cells, neurons and blood cells (39,40). Adults have a small number of stem cells when compared to a developing embryo and the stem cell of an embryo more easily obtained (41). Human embryonic stem cells (hES) derived from the inner mass of the preimplantation blastocyst have the potential to differentiate into all cell types in the human body and thus are valued by researchers (50). However, due to ethical and political issues, the obtaining and experimentation on human embryonic stem cells is difficult and alternate means are needed as stem cell provides potentially new treatments for a range of diseases (54).
Rabbits can be used both in the obtaining of stem cell for experimentation and also as recipients of stem cells to cure diseases. The human-rabbit hybrid embryo provides a method of obtaining stem cells for experimentation and these stem cells possess many similar properties to human stem cells, including expression of surface markers, special growth requirements, capabilities of self renewal, formation of embryonic body and differentiation into cells of all three germ layers (53).
Stem cell experimentation on rabbits also focuses on transplanting stem cell into rabbits to treat specific diseases. A study at Zhejiang University where a research team transplanted human mesenchymal stem cell into New Zealand white rabbits with myocardial infarction recoded regeneration of vascular structure and improvement in cardiac function. The research team noted the potential of stem cells in differentiating into specialized cardiac cells when subjected to the specific conditions of the rabbit heart (52).
Another study at Hallym University tested the effectiveness of transplanting mesenchymal stem cells from New Zealand white rabbits into damaged cartilage in the knees of other New Zealand white rabbits. In the experiment, mesenchymal stem cells were obtained from the rabbit bone marrow and injected into the knees of other rabbits that had cartilage in their knees surgically damaged. The research team were able to conclude notable cartilage recovery in the rabbits with the mesenchymal stem cell injection and the potential of mesenchymal stem cells to differentiate into fibroblasts, adipoblasts, osteoblasts and chondroblasts (51,53).
The findings in stem cell experimentation with in rabbits yielded positive results and potentially opens a new field of research in the use of stem cell to treat a range of diseases in humans.
- Dr Mark Hill 2009, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G 
- E.Horne Craigie, University of Toronto Press 1948, Bensley's Practical Anatomy of the Rabbit 8th Edition
- Ankum WM, Houtzager HL, Bleker OP (1996). "Reinier De Graaf (1641-1673) and the fallopian tube". Human Reproduction Update 2 (4): 365–9. doi:10.1093/humupd/2.4.365. PMID 9080233.
- Jocelyn HD, Setchell BP (December 1972). "Regnier de Graaf on the human reproductive organs. An annotated translation of Tractatus de Virorum Organis Generationi Inservientibus (1668) and De Mulierub Organis Generationi Inservientibus Tractatus Novus (1962)". Journal of Reproduction and Fertility. Supplement 17: 1–222.
- Jay, V. (2000). "A portrait in history. The legacy of Reinier de Graaf". Archives of Pathology & Laboratory Medicine 124(8): 1115–6.
- Lois N Magner. "A history of the Life Sciences" 3rd edition
- J. D. Biggers. (1991). Walter Heape, FRS: a pioneer in reproductive biology.Centenary of his embryo transfer experiments Laboratory of Human Reproduction and Reproductive Biology, and Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, MA 02115, USA
- National Center for Biotechnology Information. (June 16, 2009). Rabbit Genome Resources, Retrieved August 20, 2009, from NCBI Rabbit Genome Site
- The Broad Institute. (2008). Rabbit Genome Sequencing Project. Retrieved only August 29, 2009, from Rabbit Genome Sequencing Project
- Campbell, M. A. (2002). Sequencing Whole Genomes: Hierarchical Shotgun Sequencing v. Shotgun Sequencing Retrieved August 31, 2009, from Shotgun Sequencing - 1
- Trivedi, B. (2000). Sequencing the Genome. Retrieved September 4, 2009, from Shotgun Sequencing - 2
- Mage, R. (2008). Rabbit genome sequencing update: genes of immunological interest found in the 2x genome assemblies, ENCODE, and the 7x trace archive. Journal of FASEB 22(Retrieved September 5, 2009, from Rabbit Genome & its Immunological Interest
- Ensembl (2009). Rabbit (Oryctolagus cuniculus). Retrieved September 5, 2009, from Ensembl Genome Project
- Korstanje, R. et al. (1999) Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting. Cytogenetics and cell genetics 86(3-4), 317-322. Retrieved September 6, 2009, from Complete homology maps of the rabbit Article "PMID 10575232"
- Brunner, R., Knopp, A., Rudolph, W. (n.d). Rabbit chromosome analysis by image processing. Journal of Applied Rabbit Research.
- DebMark Rabbit Education Resource (2006). Rabbit Genetics. Retrieved September 6, 2009, from Rabbit Genetics
- Painter, T. (2009) Studies in mammalian spermatogenesis VI. The chromosomes of the rabbit. Journal of Morphology. 43(1): 1-43.
- Hofsaess, F., and Meacham, T. ( 1995) Chromosome abnormalities of early rabbit embryos. Journal of Experimental Zoology. 177 (1): 9-11 Chromosome abnormalities
- Martin-Deleon, P., Shaver, E., and Gammal, E. (1973) Chromosome abnormalities in rabbit blastocysts resulting from spermatozoa aged in the male tract. Fertility and Sterility. 24(3):212-219.
- Patil, M. et al (2004) Chromosome analysis of domestic rabbit. The Journal of Bombay Veterinary College. 12 (1 and 2)
- Biggers J.D (1991) Walter Heape FRS: a pioneer in reproductive biology. centenary of his embryo transfer experments.Journals of reproduction and fertility. 93(1)
- Stoodley, M (2006). Hydrocephalus. Prince of Wales Clinical School. Retrieved from 
- National Institute of Neurological Disorders and Stroke (2008, February). Hydrocephalus Fact Sheet. Retrieved from 
- Lewin, R (1999). Is Your Brain Really Necessary?. 24) Developmental Medicine & Child Neurology, 41. Retrieved from 
- Bigio, M.R, & Bruni J.E (1991). Silicone oil-induced hydrocephalus in the rabbit . Child's Nervous System, 7(2), Retrieved from 
- Geelen, J.A (1974). A case of hydrocephalus and meningoencephalocele in a rabbit, caused by aqueductal malformation. Laboratory Animals, 8(2), Retrieved from 
- LAMMINGX, G.E (1954). 27) Hydrocephalus in Young Rabbits Associated with Maternal Vitamin A Deficiency. British Journal of Nutrition, 8(2), Retrieved from 
- Carney, A (2008, February ). Spina bifida. Retrieved from 
- Better Health Channel (2008, October ). Spina bifida explained . Retrieved from 
- Peeters, M.C, Viebahn, C, & Hekking, J.W (1998). Neurulation in the rabbit embryo . Anatomy and Embryology, 197(3), Retrieved from 
- George, T.M, & Fuh E (2003). Review of Animal Models of Surgically Induced Spinal Neural Tube Defects: Implications for fetal surgery. Pediatric Neurosurgery , 39(2), Retrieved from 
- Pedreira, D.A, & Valente P.R (2002). A Different Technique to Create a 'Myelomeningocele-Like' Defect in the Fetal Rabbit. Fetal diagnosis and therapy, 17(6), Retrieved from 
- Patten, B.M (1953). Embryological stages in the establishing of myeloschisis with spina bifida. The American journal of anatomy, 93(3), Retrieved from 
- Pedreira, D.A, & Valente P.R (2003). Successful Fetal Surgery for the Repair of a 'Myelomeningocele-Like' Defect Created in the Fetal Rabbit. Fetal Diagnosis and Therapy, 18. Retrieved from 
- Lecklitner, M.L, & Douglas K.P (1986). Osteoscintigraphy and brachydactylia of the hand . European Journal of Nuclear Medicine and Molecular Imaging, 12(9), Retrieved from 
- Greene, H.S, & Saxton J.A (1938). HEREDITARY BRACHYDACTYLIA AND ALLIED. ABNORMALITIES IN THE RABBIT. THE JOURNAL OF EXPERIMENTAL MEDICINE , 69. Retrieved from 
- P. Ianakiev, M. van Baren, M. Daly, S. Toledo, M. Cavalcanti, J. Neto, E. Silveira, A. Freire-Maia, P. Heutink, M. Kilpatrick. Acheiropodia Is Caused by a Genomic Deletion in C7orf2, the Human Orthologue of the Lmbr1 Gene. The American Journal of Human Genetics, 68. Retrieved from 
- Walbaum, R, Hazard, C, & Cordier, R (1976). Brachydactylia with symphalangism, probably autosomal recessive . Human Genetics, 33(2), Retrieved from 
- Campbell, N.A, & Reece, J.B (2002). Biology 6th Edition. USA: Benjamin Cummings.
- Solomon, E.P, Berg, L.R, & Martin, D.W (2002). Biology 6th Edition. USA: Thompson Learning.
- Glanville, A.R (2008). Scientifica. NSW, Australia: Millennium House.
- Lund, R.D (2008). Saving Vision with Skin Cells. Retrieved from 
- Jones, B.W, & Marc R.E (2008). Computational Molecular Phenotyping and Excitation Mapping in the P347L. Retinal Degeneration , (2986), Retrieved from 
- Kondo, M, Sakai, T, & Ueno, S (2009). Generation of a Transgenic Rabbit Model of Retinal Degeneration. Investigative Ophthalmology and Visual Science, 50. Retrieved from 
- Punzo, C, Chen, B, & Roesch, K (2009). PHOTORECEPTOR DEATH IN MOUSE MODELS OF RETINITIS PIGMENTOSA. Retrieved from 
- Zimmer, M (2008). Alba. Retrieved from 
- Scott, C (2006, May 1). Chimeras in the crosshairs. Nature Biotechnology , 24, Retrieved from 
- Mott, M (2005, January 25). Animal-Human Hybrids Spark Controversy. National Geographic News, Retrieved from 
- Li, S, & Chen X (2006). Rabbits generated from fibroblasts through nuclear transfer . Society for Reproduction and Fertility, 131. Retrieved from 
- Minger, S (2007). Interspecies SCNT derived human embryos. Regenerative Medicine, 2(2), Retrieved from 
- Koga, H, Shimaya, M, & Muneta, T (2008). Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect. Arthritis Research & Therapy, 10(4), Retrieved from 
- Wang, J, Li, C, & Sun, Y (2005). Human bone marrow-derived mesenchymal stem cells transplanted into damaged rabbit heart to improve heart function. Journal of Zhejiang University Science, 6(4), Retrieved from 
- Chen, Y, Liu, A, & Mao, W (2003). Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Research , 13(4), Retrieved from 
- Bethesda (2009, September 23). Stem Cell Information. Retrieved from 
- Cibelli, J. B. (2002). Principles of cloning Amsterdam. Academic Press
- European rabbit (oryctolagus cuniculus) Retrieved 9/21/2009, 2009, from http://www.feral.org.au/content/species/rabbit.cfm
- Warner, S. (2003). Inositol transport in preimplantation rabbit embryos: Effects of embryo stage, sodium, osmolality and metabolic inhibitors Reproduction, 125(4), 479-493.
- Derelanko, M. J. (2008). The toxicologist's pocket handbook, second edition Informa HealthCare
- Beaudoin, S., Barbet, P., & Bargy, F. (2003). Developmental stages in the rabbit embryo: Guidelines to choose an appropriate experimental model Fetal Diagnosis and Therapy, 18(6), 422-427.
Anesthetic - A drug that causes temporary loss of bodily sensations
Aplasia - The absence or defective development of a tissue or organ
Blastocyst - A stage of embryo development that occurs about five days after fertilisation when the embryo contains quite a few cells
Caudal - Situated towards the inferior or posterior end of the body
Cephalic - Relating to the head
Cerebral Aqueduct - A canal filled with cerebrospinal fluid within the midbrain
Cerebral Cortex - The layer of unmyelinated neurons (the grey matter) forming the cortex of the cerebrum of the brain
Cerebro Spinal Fluid (CSF) - A watery fluid which flows in the cavities within the brain and around the surface of the brain and spinal cord
Chromosome - Microscopic carriers of genetic material, composed of deoxyribonucleic acid (DNA) and proteins and appearing as rods under a microscope
Cisterna Magna - is one of three principal openings in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain
Diploid - A full set of genetic material, consisting of paired chromosomes one chromosome from each parental set
Dorsal - Position towards the back
Fontanel - Membranous gap between the bones of the cranium in an infant or fetus
Genome - All genetic information, the entire genetic complement and all of the hereditary material possessed by an organism. Made up of both chromosomal genome (inside the nucleus of the cell in the familiar form of chromosomes) and mitochondrial genome (outside the nucleus in the cytoplasm of the cell, usually in the form of one round chromosome (the mitochondrial chromosome))
Genotype - The genetic makeup of an organism
Glucocorticoids - A class of steroid hormones that bind to the glucocorticoid receptor (GR), which is present in almost every vertebrate
Haploid - A single set of chromosomes (half the full set of genetic material)
Laparotomy- Surgical incision into the abdominal wall; often done to examine abdominal organs
Ligases - Group of enzymes that catalyze the binding of two molecules
Oocyte - A female gametocyte that develops into an ovum after two meiotic divisions
Phenotype - The observable traits or characteristics of an organism, for example hair color, weight, or the presence or absence of a disease.
Pronuclear - Haploid nucleus before fusion of nuclei in fertilisation
Restriction Endonucleases - Enzymes that recognize and cleave specific DNA sequences, generating either blunt or single-stranded (sticky) ends
Retina - The thin layer of cells at the back of the eyeball where light is converted into neural signals sent to the brain
Rostral - Situated toward the oral and nasal region
Somite - Blocks of mesoderm on either side of the notochord and neural tube during development of the vertebrate embryo. Develop into muscles and vertebrae
Stenosis - Abnormal narrowing of a bodily canal or passageway
Weaning - Young become accustom to nourishment other than suckling
Links to Research Laboratories and Researchers
1) Transgenic lab: 
Involved in development of transgenic organisms, DNA construction and phenotype analysis.
2) World Rabbit Science Association: (WRSA) 
International Association where researchers exchange knowledge and encourage teaching, scientific research, practical experimentation, the collection and publication of statistics and documents relating to the rabbit. Even have their own journal called “World Rabbit Science”
Website provides contact with researchers, professors and veterinarians working with rabbits.
Example: Dr. Myriam Kaplan-Pasternak, Doctorate Veterinary Medicine, University of California, firstname.lastname@example.org
3) American Veterinary Medical Association 
The American Veterinary Medical Association (AVMA), established in 1863, is a not-for-profit association representing more than 78,000 veterinarians working in private and corporate practice, government, industry, academia, and uniformed services. Excellent website into animal research and has a range of veterinary journals.