29 May 2009

With an unobstructed view and just the right atmospheric conditions, look to the west at sunset and you might catch a glimpse of the famously fleeting green flash. For the right conditions to generate sustainable transgenic primate models of human neurodegenerative diseases, look to the east. The debut of three green-glowing marmosets in the 28 May Nature suggests that such models could soon be on the horizon. Researchers in Japan led by Erika Sasaki at the Central Institute for Experimental Animals, Kawasaki, and Hideyuki Okano at Keio University School of Medicine, Tokyo, have generated common marmosets that carry a transgene for enhanced green fluorescent protein (EGFP). That transgene has been passed from one of the monkeys to his offspring—a milestone in primate transgenics. The work raises the prospects of colonies of marmosets that express genes that cause Alzheimer’s, Parkinson’s, or other neurodegenerative diseases that are incompletely modeled by transgenic mice. But as a Nature editorial points out, the work will undoubtedly raise concerns about animal welfare and may even fuel debate over human transgenics. There are also questions about the suitability of New World marmosets to model human diseases since they are even more distantly related to us than Old World primates, such as rhesus macaques. “The value of transgenic marmosets is still an empirical question that ultimately will be answered by a comprehensive analysis of animals expressing disease-related transgenes,” suggested Larry Walker, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, in an e-mail to ARF.

This is not the first transgenic primate. Transgenic macaques have been glowing since Anthony Chan, Gerald Schatten, and colleagues at the Oregon Regional Primate Center, Beaverton, introduced the GFP gene into their genomes in 2001 (see Chan et al., 2001), and just last year Chan, now at Yerkes, successfully generated macaques carrying the huntingtin gene (see Yang et al., 2008). But none of those monkeys have passed their transgenes on to the next generation.

Sasaki and colleagues used a similar approach to the macaque work, using lentiviruses to insert the EGFP gene into marmoset embryos. The researchers opted to transfect the transgene into natural embryos, remove, then re-implant them into the females after lentiviral injection. This procedure seems to have a slightly better success rate than using in-vitro fertilized (IVF) embryos. Every transfected natural embryo expressed the transgene, whereas only 70 percent of transfected IVF embryos did.

Eighty transfected embryos were implanted into 50 surrogate mothers, of which seven became pregnant. Three miscarried and four carried their babies to term and delivered successfully. One mother had twins. Three of the five babies glowed green under ultraviolet light and tissue samples collected non-invasively (placenta, hair roots, skin, and peripheral blood cells) showed various levels of EGFP expression. Southern blotting showed that copies of the transgene had been integrated into numerous genomic sites in the different animals. One female (No. 584) had at least four copies of the gene on three different chromosomes, while a male (No. 666), also called Kou, had up to 13 different integration patterns. It is not clear if the three miscarriages resulted from lethal insertion of the gene into the genome.

When the animals reached sexual maturity the researchers tested their gametes for the EGFP gene. Semen samples showed that Kou’s spermatozoa expressed the transgene, while one of three live natural embryos from female 584 also had strong EGFP expression. Sasaki and colleagues used Kou’s sperm for IVF and found that 20-25 percent of the embryos strongly expressed EGFP. Three of those embryos were implanted into a surrogate mother and one baby was carried to term. That baby expressed the transgene in the skin, but not in the placenta or in the hair.

“The birth of this transgenic baby is undoubtedly a milestone,” write Schatten, now at the University of Pittsburgh School of Medicine, Philadelphia, and Shoukhrat Mitalipov from the Oregon National Primate Research Center in Beaverton, in an accompanying Nature News & Views. “Subsequent generations can be produced by natural propagation, with the eventual establishment of transgene-specific monkey colonies—a potentially invaluable resource for studying incurable human disorders, and one that may also contribute to preserving endangered primate species,” they write.

In a Nature press briefing, Okano indicated that he hopes to use the technology to develop marmoset models of Parkinson disease and amyotrophic lateral sclerosis. But the researchers hinted that the next steps will not be straightforward due to certain limitations of the methodology. One is that the selection method depends on embryonic expression, which precludes the use of promoters that target transgene expression to specific tissues, such as neurons in the brain. The other major limitation is that the transgene must be kept below about 10 Kb of DNA, which is smaller than some amyloid precursor protein constructs (see Borchelt et al., 1996). And to date, transgenes cannot be targeted into specific locations in the genome, as with homologous recombination.

The marmosets themselves may pose some limitations, as well. Though these animals are quite prolific, a single female having up to 80 babies, as opposed to 10 for macaques, they are more distantly related to humans. It is not clear that they exhibit the same kind of cognitive deficits that characterize Alzheimer disease (AD), though they do appear to spontaneously develop amyloid deposits (see Maclean et al., 2000). Okano told ARF via e-mail that he is currently working with collaborators to develop behavioral tests to measure cognitive function in marmosets and that he is interested in studying age-related cognitive decline in the monkeys.

“The advantages are that they are small (about 250 g) but with relatively large brains (about 8 g), and they have been used in some significant studies of neurodegenerative conditions,” Walker told ARF. But a disadvantage might be their longevity. “They can live to around 16 years in captivity, so the development of phenotypes associated with senescence could be much slower than in transgenic mice, he suggested. That could make studies prohibitively expensive. Okano estimates that a program using transgenic marmosets in preclinical trials would cost more than 100 times that of a similar program using transgenic mice.—Tom Fagan

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References

Paper Citations

1. Chan AW, Chong KY, Martinovich C, Simerly C, Schatten G. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science. 2001 Jan 12;291(5502):309-12. PubMed.
2. Yang SH, Cheng PH, Banta H, Piotrowska-Nitsche K, Yang JJ, Cheng EC, Snyder B, Larkin K, Liu J, Orkin J, Fang ZH, Smith Y, Bachevalier J, Zola SM, Li SH, Li XJ, Chan AW. Towards a transgenic model of Huntington's disease in a non-human primate. Nature. 2008 Jun 12;453(7197):921-4. PubMed.
3. Borchelt DR, Davis J, Fischer M, Lee MK, Slunt HH, Ratovitsky T, Regard J, Copeland NG, Jenkins NA, Sisodia SS, Price DL. A vector for expressing foreign genes in the brains and hearts of transgenic mice. Genet Anal. 1996 Dec;13(6):159-63. PubMed.
4. Maclean CJ, Baker HF, Ridley RM, Mori H. Naturally occurring and experimentally induced beta-amyloid deposits in the brains of marmosets (Callithrix jacchus). J Neural Transm. 2000;107(7):799-814. PubMed.

Further Reading

News

1. Chimpanzees Get AD Pathology—But Clock Is Ticking on Research 28 May 2008
2. Monkey Genetics: Transgenic Rhesus With Huntington Disease 21 May 2008