Molecular Biology Animal

Guolong Zhang

Oklahoma State University, Stillwater, Oklahoma, U.S.A.

INTRODUCTION

The past two decades have brought a revolution in molecular biology research and the emergence of new techniques such as high-throughput DNA sequencing, microarray, nuclear transfer, RNA interference, and mass spectrometry-based proteomic techniques. With these new technologies it is now possible to sequence the entire genome of any animal species, carry out systematic genome-wide screens of gene functions, and manipulate (delete, mutate, overexpress, or suppress) virtually any individual gene in the genome. Such new tools are now being applied to most aspects of animal production and will undoubtedly have a profound impact on animal agriculture by improving production efficiency and sustainability, animal health, food safety, and environmental stability. This article briefly describes the major molecular techniques and their application in animal agriculture.

NEW EMERGING MOLECULAR BIOLOGY TECHNIQUES

High-Throughput DNA Sequencing

With the advent of automated DNA sequencing technology in the mid-1980s, which involves the labeling of DNA fragments with fluorescent dyes, sequencing the entire genome has become a reality, leading to the recent explosion in genomics research.[1] Following the completion of the human genome in early 2003, nearly 200 genomes of mammals, plants, insects, and microbes, as well as over 1200 viral genomes, have been sequenced so far. The dog and chicken genomes have been sequenced at a coverage of one-and-a-half and sevenfold, respectively, while the large-scale genome sequencing of other major farm animal species, including cattle, pig, horse, and aquatic species, is either in progress or will soon be initiated. Complementary DNA (cDNA) sequences, also known as expressed sequence tags (EST), from hundreds of cell/tissue-specific libraries of farm animals have also been sequenced.[2-4] The availability of such enormous amounts of genomic and cDNA sequence information will undoubtedly facilitate the identification of genes of agricultural importance and development of new strategies for improving production efficiency of livestock.

High-Throughput Differential Gene Expression Techniques

Analysis of the changes in gene expression pattern associated with biological processes is critical to understand gene function. The recent development of powerful high-throughput differential gene expression techniques allows simultaneous detection of differential expressions of tens of thousands of genes in a single experiment1-5-1 (Table 1). Among commonly used techniques, DNA microarray has attracted tremendous interest among biologists since its introduction in 1995.[2-5] Utilizing a simultaneous two-color hybridization scheme, DNA microarray promises sensitive, quantitative monitoring of gene expression profiles on the whole genome scale. Such a tool has been applied to farm animal research for studying reproduction, muscle development, fat deposition, nutrient utilization, and disease resistance for all major livestock species.[2-4] Hundreds of putatively important genes have been identified and their biological functions are being confirmed.

Transgenesis

Gene(s) of interest can be modified and reintroduced into animals to create new breeds with desired traits by the technique of transgenesis. The major strategies to generate transgenic animals have gone from pronuclear injection, embryonic stem cells, and nuclear transfer to the latest lentiviral delivery.[6] Nuclear transfer, a cloning technology introduced in 1996 that involves the injection of nuclei of genetically modified somatic cells into the enucleated oocytes, allows precise targeting of genomes in virtually any animal species. Transgenic animals from all major livestock species have been generated by nuclear transfer, but this technique suffers from low efficiency and high technical demands.[6] The lentiviral delivery of transgenes is extremely efficient in introducing foreign gene(s) into the germline by taking advantage of the ability of lentivirus to integrate into both dividing and

Table 1 Comparison of major high throughput differential gene expression techniques

Minimum

Simultaneous

Simultaneous

Detection of

Require previous knowledge of

Detection of

mRNA requirement

comparison of >2 samples?

detection of > 2 genes?

quantitative difference?

mRNA or gene sequences?

unknown genes?

ESTa

0.01 1 mg

Yes

Yes

Yes

No

Yes

sequencing cDNA RDA

0.01 0.1 mg

No

Yes

No

No

Yes

Differential

0.01 0.1 mg

Yes

Yes

Yes

No

Yes

display SSH

0.5 1 mg

No

Yes

No

No

Yes

SAGE

1 5 mg

Yes

Yes

Yes

No

Yes

DNA

0.1 1 mg

Yes

Yes

Yes

Yes

No

microarray Real time

1 cell

Yes

Yes, but limited

Yes

Yes

No

RT PCRb

Abbreviations: EST, expressed sequence tag; cDNA RDA, complementary DNA representational difference analysis; SSH, suppression subtractive hybridization; SAGE, serial analysis of gene expression; RT PCR, reverse transcriptase PCR; mRNA, messenger RNA.

bStrictly speaking, real time RT PCR is not a high throughput technique, but provides a highly sensitive and quantitative analysis of gene expression, and is therefore listed here for comparison.

Abbreviations: EST, expressed sequence tag; cDNA RDA, complementary DNA representational difference analysis; SSH, suppression subtractive hybridization; SAGE, serial analysis of gene expression; RT PCR, reverse transcriptase PCR; mRNA, messenger RNA.

bStrictly speaking, real time RT PCR is not a high throughput technique, but provides a highly sensitive and quantitative analysis of gene expression, and is therefore listed here for comparison.

nondividing cells.[6] Therefore, it appears to be economically possible for genetic manipulation of livestock in the future.

RNA Interference

RNA interference emerged in 1998, following an observation that the introduction of double-stranded RNA into cells induces potent and specific gene silencing.[7] It has become the method of choice for analysis of gene functions, particularly in mammalian systems. Combined with genomics data, RNA interference could allow functional determination of any gene encoded in the genome. In animal agriculture, it holds great promise for use against infections by expressing small interfering RNA to disrupt the replication of pathogens or to inhibit the expression of receptors for pathogens. In addition, germline integration of interfering RNA by the lentiviral delivery technique could generate new animal strains with enhanced disease resistance or suitable organs for xenotransplantation. Such an application is expected to be seen within 5 years.

Proteomic Techniques

All biological processes ranging from development to reproduction, response, and infection are ultimately dependent upon the selective expression and interactions of a complex network of proteins. Major proteomic techniques include two-dimensional polyacrylamide gel electrophoresis, yeast two-hybrid, proteome microarray, and various formats of mass spectrometry.[8] These techniques allow detecting expression levels, interactions, posttranslational modifications, or enzymatic activities of thousands of proteins simultaneously. Most of these tools have yet to be utilized in animal agriculture, but are expected to have a significant impact in the next 5 10 years.

APPLICATION OF MOLECULAR BIOLOGY IN ANIMAL AGRICULTURE

Animal Growth and Production Efficiency

A number of genes associated with growth and productivity traits have been identified by genome-mapping approaches.[1,9] The availability of these genes provides an excellent opportunity for marker-assisted breeding[9] and genetic manipulation by transgenesis[6] to produce new breeds with desired traits. Generation of transgenic animals bearing the desired genes or gene alleles is the most straightforward approach. For example, transgenic pigs and sheep overexpressing growth hormone or growth hormone-releasing factor normally grow faster and utilize feed more efficiently than nontransgenic littermates.[6,10] Transgenesis efforts to alter milk composition, wool formation, and meat quality are currently ongoing. With the advances of new molecular techniques, particularly through genome sequencing and DNA microarray, additional genes will be identified, providing more and better targets for breeding and genetic manipulation.

Animal Health and Well-Being

Genome sequencing of veterinary and zoonotic pathogens has led to a better understanding of microbial pathogenesis and development of novel approaches for producing safer and more effective vaccines. Differential gene expression techniques are being employed for comprehensive studies of microbial pathogenesis and the mechanisms of host defense.[11] It is expected that important genes involved in microbial pathogenesis or host immune responses will be identified for all major veterinary pathogens. Enhanced disease resistance can be and has been demonstrated in farm animals by disruption of certain disease-susceptible genes (such as Prion protein and pathogen receptors) or by overexpression of immunomodulatory cytokines, microbial antigens, and immu-noglobin genes specific for certain pathogens.[6,10] Transient viral delivery of such immune-responsive genes into animals at certain production stages (e.g., postweaning and onset of disease) also appears to be a viable approach. Rapid disease diagnosis and monitoring by differential expression techniques, particularly realtime polymerase chain reaction (RT-PCR), is another notable application.

Biomedical Applications

The use of transgenic farm animals as bioreactors for producing pharmaceuticals and as organ donors for transplantations in humans has attracted vast attention in the past two decades. A few therapeutic proteins, such as a-1 antitrypsin and antithrombin III, have been produced in large quantities in the milk or blood of large farm animals,[6,10] and are currently in advanced phases of clinical trials. Because of a worldwide shortage of human organs available for transplantation, pigs are being explored for their potential as organ donors. In order to reduce hyperacute rejection of pig transplants, several genes associated with this process, such as a-1,3-galactosyltransferase, have been deleted and knockout pigs generated.[6,10] Availability of such transgenic pigs is expected to have important applications in xenotransplan-tation. In addition, development of transgenic farm animals as experimental models for human diseases that cannot be recapitulated in rodents is another attractive and important application.

Environmental and Other Applications

Modern intensive animal production practice poses considerable stress to the environment by creating significant amounts of animal waste. Molecular strategies to reduce the pollution are also being sought. Recently, transgenic pigs expressing a bacterial phytase gene were shown to have improved uptake of organic phosphate, thus reducing the need for inorganic phosphate supplementation in feed and release of phosphorus in manure.[10] New strains of microbes have also been genetically engineered with enhanced ability to convert animal waste into less polluted material. Another potential application includes the generation of new exotic breeds of pets for leisure purposes. GloFish, a transgenic zebra fish expressing fluorescent protein, is a notable example that is currently on sale in the United States (http://www. glofish.com/).

CONCLUSION

These newly emerged molecular techniques are beginning to transform the research and practice of animal agriculture, and will be instrumental in developing profitable, environment-friendly agriculture and in meeting the demands of growing world populations. The complete genomic sequences and DNA microarrays featuring the whole genome of major farm animal species are expected to be available within the next 5 10 years. Novel approaches to enhancing animal productivity and health will be developed, and new biotherapeutics and new animal breeds with desired characteristics will be produced. In addition, transgenic farm animals hold great promise to provide much needed pharmaceuticals and immunologically suitable organs for transplantation. However, it should be kept in mind that usage of transgenic animals for the production of foods and pharmaceuticals and for xenotransplantation will require extensive characterization before release to the market. It is our scientists' responsibility to ensure the safety of genetically modified animals and animal products, and also to educate the general public for its increased acceptance of animal biotechnology.

ACKNOWLEDGMENTS

I would like to thank Drs. Wilson Pond, Joan Lunney, Rodney Geisert, and Udaya DeSilva for their thoughtful suggestions and critical review of the manuscript. I apologize for failing to refer to all primary sources due to space constraints.

REFERENCES

1. Rohrer, G. Genomics. In Encyclopedia of Animal Science; Pond, W.G., Bell, A.W., Eds.; Marcel Dekker, Inc.: New York, 2004.

2. Suchyta, S.P.; Sipkovsky, S.; Kruska, R.; Jeffers, A.; McNulty, A.; Coussens, M.J.; Tempelman, R.J.; Halgren,

R.G.; Saama, P.M.; Bauman, D.E.; Boisclair, Y.R.; Burton, J.L.; Collier, R.J.; DePeters, E.J.; Ferris, T.A.; Lucy, M.C.; McGuire, M.A.; Medrano, J.F.; Overton, T.R.; Smith, T.P.; Smith, G.W.; Sonstegard, T.S.; Spain, J.N.; Spiers, D.E.; Yao, J.; Coussens, P.M. Development and testing of a high density cDNA microarray resource for cattle. Physiol. Genomics 2003, 15 (2), 158 164.

3. Tuggle, C.K.; Green, J.A.; Fitzsimmons, C.; Woods, R.; Prather, R.S.; Malchenko, S.; Soares, B.M.; Kucaba, T.; Crouch, K.; Smith, C.; Tack, D.; Robinson, N.; O'Leary, B.; Scheetz, T.; Casavant, T.; Pomp, D.; Edeal, B.J.; Zhang, Y.; Rothschild, M.F.; Garwood, K.; Beavis, W. EST based gene discovery in pig: Virtual expression patterns and comparative mapping to human. Mamm. Genome 2003, 14 (8), 565 579.

4. Cogburn, L.A.; Wang, X.; Carre, W.; Rejto, L.; Porter, T.E.; Aggrey, S.E.; Simon, J. Systems wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes. Poult. Sci. 2003, 82 (6), 939 951.

5. Liang, P.; Pardee, A.B. Analysing differential gene expression in cancer. Nat. Rev. Cancer 2003, 3 (11), 869 876.

6. Clark, J.; Whitelaw, B. A future for transgenic livestock. Nat. Rev. Genet. 2003, 4 (10), 825 833.

7. Hannon, G.J.; Conklin, D.S. RNA interference by short hairpin RNAs expressed in vertebrate cells. Methods Mol. Biol. 2004, 257, 255 266.

8. Zhu, H.; Bilgin, M.; Snyder, M. Proteomics. Annu. Rev. Biochem. 2003, 72, 783 812.

9. Thallman, M. Selection: Marker Assisted. In Encyclopedia of Animal Science; Pond, W.G., Bell, A.W., Eds.; Marcel Dekker, Inc.: New York, 2004.

10. Niemann, H.; Kues, W.A. Application of transgenesis in livestock for agriculture and biomedicine. Anim. Reprod. Sci. 2003, 79 (3 4), 291 317.

11. Munir, S.; Kapur, V. Transcriptional analysis of the response of poultry species to respiratory pathogens. Poult. Sci. 2003, 82 (6), 885 892.

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