2009 Group Project 1

Introduction

Several characteristics of the rabbit make it an excellent model for study. Many studies have resulted in the development and improvement of various micro-manipulation techniques such as the production of transgenic rabbits. The rabbit is an appropriate animal model as the results from many experiments are significant to that of other mammals, including humans.

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 upto 30 to 40 young in this time.

Staging

Table 1. Carnegie classification for stages of rabbit development

Stage	Days	Event
10	8.5	First somites appear, rostral neuropore closes
12	9.5	Rostral limb bud, embryonic pedicle, caudal neuropore closes, one cerebral vesicle, pharyngeal arches, completion of optic vesicle
13	10.5	Dosral C-shaped curvature, caudal limb bud, prominent cardiac swelling, four pharyngeal arches
14	11.5	Three cerebral vesicles, optic plate, mesonephric ridge
15	12.5	Hand plate, wheel ray limbs, lens vesicle closes, five cerebral vesicles, facial buds, vertebrae
16	13.5	Foot plate, finger rays, prominent liver, intestinal loops, auricular hillocks
19	14.5	Cuboidal body, trunk straightens, parallel limbs, toe rays, modeled face, visible auricle
20	15.5	Elbow, fingers elongate, notches between toe rays
21-22	16.5	Toes free, cecal bud in intestinal hernia
22-23	17.5	Knee, neck, eyelids
23	18.5	Hands and feet join at the midline, closure of abdominal wall, eyelids cover eyes
Following this stage, fetal appearance is completed and organogenesis is achieved

History of Model Use

Regnier de Graaf

key discoveries in reproductive biology

He had become engaged in a priotiry battle about finding the mammalian ovum, but both had been mistaken. In 1672 Swammerdam published an account of his study of the “female testes,” incluing his claim that he had discovered the human ovum. During the same year, De Fraaf published a treatise call 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, ewes and women, he assumed they were mammalian eggs. De Fraaf also described the corpus luteum (follicles from which the mature egg had already escaped). Leeuwenhoek argued that the structures now known as Graafian follicles could not be eggs, but Haller suggested that the egg might be formed by the coagulation of the fluid in 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.

Although the rabbit embryo was not visible until about 10days of gestation, about one third of the total time of pregnancy, deGraaf’s work seemed to provide microscopic evidence that viviparous animals arise from eggs formed by the obaries and ovist preformationists assumed that his observations supported their doctrine.

Walter Heape (1855-1928)

About 100 years ago, on 27 April 1890, Walter Heape transferred rabbit embryos from one mother to another. One animal became pregnant and delivered young from the transferred embryos. Thus the first mammalian embryo transfer experiment was successfully completed. His embryo transfer work in perspective as it relates to other contributions of this pioneer in reproductive biology.

embryo transfer experiments

In 1891, Walter Heape (1855-1929), a professor and physician at the University of Cambridge, England, who had been conducting research on reproduction in numerous animal species, reported the first known case of embryo transplantation. Working with two species of rabbits, he flushed embryos from the oviducts (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.

As a result of this work, many scientists became interested in culturing eggs and embryos in the laboratory. Gregory Pincus and colleagues were the first to show how eggs of various animals would undergo maturation if released from their follicle and cultured in a laboratory.

In 1939, he reported that human eggs would mature in the laboratory within 12 hours. It was not until 1959, however, that M.C. Chang reported, in the journal Nature, the first unequivocal case of a live birth following egg fertilization in the lab, true in vitro fertilization and subsequent embryo transfer, to the uterus.

Explanation for his experiments

He asked three questions:

1.Is it possible to make use of the uterus of one variety of rabbit as a medium for the growth and complete foetal development of fertilized ova of another variety of rabbit?

2.What effect, if any, does a uterine foster-mother have upon her foster-children?

3.Does the presence and development of foreign ova in the uterus of a mother affect the offspring of that mother born at the same time?

Heape's final answers to these questions were given in his 1897 paper. He concluded:

That it is possible to make use of the uterus of one variety of rabbit as a medium for the growth and complete foetal development of fertilized ova of another variety of rabbit. That the uterine foster-mother exerts no modifying influence upon her foster-children in so far as can be tested by the examination of a single generation.

Genetics

Sequencing

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. (1)

The European rabbit (Oryctolagus cuniculus) 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. (2) 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. (3) (4) [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) "unfinished Oryctolagus cuniculus database" 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 useful for designing primers for PCR by predicting mRNA sequences and exon boundaries" (5)

Genome

The Rabbit genome was published by two groups.

1. 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
2. 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.

Rabbit Genome from the Nucleotide Data Bank

The rabbit genome was sequenced in 2005 by Ensembl and managed to produce: (6)

• 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

Mitochondrial Genome

The mitochondiral genome (mtDNA) of the rabbit was sequenced on November 14th 20066 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"

Taxonomy

The taxonomy ID number for the Rabbit is: 9986 Taxonomy Data

Chromosomes

There are 22 different chromosome pairs existing in each cell of the rabbit except the sex cells and red blood cells (9) 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. (7) Chromosome 12 was shorter than chromosomes 13 and 14. (8)

References

1. National Center for Biotechnology Information. (June 16, 2009). Rabbit Genome Resources, Retrieved August 20, 2009, from NCBI Rabbit Genome Site
2. The Broad Institute. (2008). Rabbit Genome Sequencing Project. Retrieved only August 29, 2009, from Rabbit Genome Sequencing Project
3. Campbell, M. A. (2002). Sequencing Whole Genomes: Hierarchical Shotgun Sequencing v. Shotgun Sequencing Retrieved August 31, 2009, from Shotgun Sequencing - 1
4. Trivedi, B. (2000). Sequencing the Genome. Retrieved September 4, 2009, from Shotgun Sequencing - 2
5. 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
6. Ensembl (2009). Rabbit (Oryctolagus cuniculus). Retrieved September 5, 2009, from Ensembl Genome Project
7. 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"
8. Brunner, R., Knopp, A., Rudolph, W. (n.d). Rabbit chromosome analysis by image processing. Journal of Applied Rabbit Research.
9. DebMark Rabbit Education Resource (2006). Rabbit Genetics. Retrieved September 6, 2009, from Rabbit Genetics

References

1. Dr Mark Hill 2009, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G [6]

2. E.Horne Craigie, University of Toronto Press 1948, Bensley's Practical Anatomy of the Rabbit 8th Edition

3.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.

4.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.

5.Jay V (August 2000). "A portrait in history. The legacy of Reinier de Graaf". Archives of Pathology & Laboratory Medicine 124 (8): 1115–6.

6.Lois N Magner, "A history of the Life Sciences" 3rd edition

7.J. D. Biggers, 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

ANAT2341 group projects

Project 1 - Rabbit | Project 2 - Fly | Project 3 - Zebrafish | Group Project 4 - Mouse | Project 5 - Frog | Students Page | Animal Development