Purdue developing less costly model for studying human disease

July 28, 2003

WEST LAFAYETTE, Ind. - A $1 million grant from the National Institutes of Health is helping Purdue University scientists move closer to making zebrafish the premier laboratory animal for studying human development and disease.

The goal of the three-year grant, which begins Aug. 1, is to create zebrafish in which a gene has been modified or permanently turned off, and the offspring inherit the alteration, said Paul Collodi, Department of Animal Sciences professor and senior researcher on the project. The progeny that possess the mutation are called knockouts. Once successfully produced in zebrafish, the knockouts will be used to study a gene's function.

The researchers are interested not just in making a genetic change, or mutation, in one fish, but also in its future generations, and more.

"The goal is to enable us to learn the role of specific genes in the zebrafish, which is an organism that is easy to study," Collodi said. "This information then can be applied to studying gene function that's involved in human disease and embryo development."

Currently knockouts are only possible in mice. Successfully duplicating the process in zebrafish would require much less time and money for researching gene function.

"The big problem in producing knockouts in species other than the mouse has been that it's been impossible to keep stem cells viable so that they contribute to the germ line, or eggs and sperm," Collodi said. "Now we have cells that we can grow for a long time in culture and they can still be transplanted into an embryo where they will become eggs or sperm."

The scientists discovered a way to keep altered embryonic stem cells from zebrafish alive long enough to pass on specific genetic changes. However they have not yet made a knockout using this technique. They currently are working to introduce a specific alteration into embryonic stem cells. It takes two generations to produce an adult fish with a specific genetic mutation once the change is made to the original embryo's sperm and eggs.

Zebrafish as model animals for genetic research offer a number of advantages, Collodi said.The first steps toward zebrafish knockouts came in a laboratory dish when the researchers created a line of zebrafish embryo cells that had not started forming specific tissues. Embryonic stem cells are those that eventually can become various types of cells throughout the body. In this case, the scientists are interested in the ones that become germ-line cells.

To sustain stem cells long enough so they successfully differentiate into germline cells, Collodi and his colleagues grew the cells in a layer of trout spleen cells.

"These spleen cells are making growth factors that kept our embryonic stem cells in a condition so that eventually we can produce fish that could pass on the traits we're hoping to study," Collodi said. The researchers are attempting to identify those growth factors.

The embryonic stem cells then were injected into a host embryo about four hours after fertilization, at a stage called the mid-blastula transition, which is the point when many of the genes in the embryo are switched on, Collodi said. The stem cells migrate all through the embryo, but the scientists needed to know if any had entered the sexual organs, or gonads, and started forming eggs or sperm.

"We want them to go into the germ line so when the fish is a sexually mature adult it will produce eggs and sperm carrying the same genetic mutations," he said. "The cells in the embryo that will become eggs and sperm are called primordial germ cells."

In order to determine if the stem cells contributed to the gonads, the researchers added a gene that makes a red fluorescent protein so the cells would glow red if they differentiated into germ-line cells. They did this by linking the red fluorescent protein gene to another gene that only is turned on in germ cells.

"So you can observe the whole process in a period of four days, and since the embryos are nearly transparent, you can see individual cells and follow where they go during development," he said. "It's relatively easy to identify very subtle mutant changes with the zebrafish. For example, it is easy to see if a blood vessel or a specific bone doesn't form exactly right."

In contrast to zebrafish, mice give birth several times a year to an average of 12 young, so there are fewer mice embryos. Also surgery must be performed in order to insert mice embryos back into the mother, while fish embryos are grown in a Petri dish. Additionally it costs much more to house, feed and care for the mice.

Collodi, who also is a member of the Program of Comparative Medicine, a collaboration of the Purdue Department of Animal Sciences, School of Veterinary Medicine and the Indiana University School of Medicine, is particularly interested in using the fish to study early development and muscle formation.

"In terms of function, there's a lot of similarity with humans," he said. "If you knock out a gene in zebrafish, the information garnered about the genetic function will be very applicable to mammals."

The NIH made mapping of the zebrafish genome a priority, and the task is expected to be completed soon.
-end-
The U.S. Department of Agriculture and the Illinois-Indiana Sea Grant College Program provided previous funding for Collodi's research with zebrafish.

Writer: Susan A. Steeves, 765-496-7481, ssteeves@purdue.edu

Source: Paul Collodi, 765-494-9280, pcollodi@purdue.edu

Related Web sites:
Purdue Department of Animals Sciences: http://www.ansc.purdue.edu/
Paul Collodi research: http://www.ansc.purdue.edu/faculty/collo_r.htm
NIH Zebrafish Genome project: http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

ABSTRACT

Zebrafish embryo cells remain pluripotent and germ-line competent for multiple passages in culture

Lianchun Fan 1, Jennifer Crodian 1, Xiangyu Liu 1, Annette Alestrom 2, Peter Alestrom 2 & Paul Collodi 1,3 (1-Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; 2-Department of Biochemistry, Physiology and Nutrition, Norwegian School of Veterinary Science, Oslo, Norway; 3-Corresponding Author: Department of Animal Sciences, Purdue University, 125 S. Russell St., West Lafayette, IN 47907)

Mouse embryonic stem (ES) cell lines are routinely used to introduce targeted mutations into the genome, providing an efficient method to study gene function. Application of similar gene knockout techniques to other organisms has been unsuccessful due to the lack of germ-line competent ES cell lines from non-murine species. Previously, we reported the production of zebrafish germ-line chimeras using short-term (24-hr old) primary embryo cell cultures (Ma et al., PNAS, 2001, 98, 2461-2466). Here we demonstrate that zebrafish embryo cells, maintained for several weeks and multiple passages in culture, remain pluripotent and germ-line competent. Zebrafish germ-line chimeras were generated from passage 5 and 6 cultures initiated from blastula- and gastrula-stage embryos. In addition to the germ line, the cultured cells contributed to multiple tissues of the host embryo including muscle, liver, gut and fin. Chimeras were also produced using embryo cell cultures derived from a transgenic line of fish that expresses the red fluorescent protein (RFP) under the control of the primordial germ cell (PGC)-specific promoter, vasa. Tissue-specific expression of RFP was detected in the gonad of the chimeric embryo. The germ-line competent embryo cell cultures will be useful for the development of a gene targeting strategy that will increase the utility of the zebrafish model for studies of gene function.

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