Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling

Abstract

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Flexible auditory training, psychophysics, and enrichment of common marmosets with an automated, touchscreen-based system

“…However, investigations into auditory capabilities and cognition increase in scope as NHPs have become genetically tractable organisms 1 , 2 , 16 – 18 . Notably, the common marmoset ( Callithrix jacchus ) has become a valuable model for biomedical research in general and the neurosciences in particular 19 – 21 .…”

Performance of Marmoset Monkeys as Embryo Donors Is Reflected by Different Stress-Related Parameters

“…When conducting animal experimental studies, it is required to reduce the amount of invasively obtained samples such as blood (number of samples and volume) to what is necessary to achieve the purpose of the study. The current study was performed with samples that were available as residual samples of scientific work related to the isolation of preimplantation embryos for embryonic stem cell line derivation and the genetic modification of marmosets [ 13 , 21 ]. Therefore, often only small numbers of samples from animals from both groups (STG and LTG) were available to be meaningfully compared.…”

“…In the last few years, the genetic modification of common marmosets ( Callithrix jacchus ) [ 9 ], a small and easy-to-handle NHP species, has attracted considerable attention, and further established the use of this species in animal models in biomedical research areas, such as neurobiology [ 10 ], reproductive biology [ 11 ], and stem cell research [ 12 ]. Genetic modification requires the application of artificial reproductive technologies (ART), such as the collection of oocytes for in vitro fertilization, or the isolation of naturally fertilized embryos for the subsequent application of gene-editing tools [ 13 ], such as CRISPR/Cas [ 14 ]. Moreover, ART procedures can be performed repeatedly in marmosets, but each time requires potentially stressful general anesthesia.…”

Generation of marmoset primordial germ cell–like cells under chemically defined conditions

“…We focussed on the marmoset Callithrix jacchus (cj), a small New World monkey, which has a short generation time compared with other NHPs, a larger litter size (two to three pups instead of singletons in macaques), and a higher gestation frequency (2 litters per year instead of one every 2–3 yr in macaques) ( Mansfield, 2003 ; Pittet et al, 2017 ). In addition, in vitro fertilisation and transgenesis technologies are well established for this species ( Sasaki et al, 2009 ; Okano et al, 2012 ; Takahashi et al, 2014 ; Drummer et al, 2021 ). We show that post-migratory PGCs in marmoset gonads express PGC genes known to be expressed also in human PGCs (hPGCs).…”