Methyl-CpG binding protein 2 (MeCP2) has crucial roles in transcriptional regulation and microRNA processing1, 2, 3, 4. Mutations in the MECP2 gene are found in 90% of patients with Rett syndrome, a severe developmental disorder with autistic phenotypes5. Duplications of MECP2-containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders6. Although Mecp2-null mice recapitulate most developmental and behavioural defects seen in patients with Rett syndrome, it has been difficult to identify autism-like behaviours in the mouse model of MeCP2 overexpression7, 8. Here we report that lentivirus-based transgenic cynomolgus monkeys (Macaca fascicularis) expressing human MeCP2 in the brain exhibit autism-like behaviours and show germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by a deep-sequencing-based method. As compared to wild-type monkeys, MECP2 transgenic monkeys exhibited a higher frequency of repetitive circular locomotion and increased stress responses, as measured by the threat-related anxiety and defensive test9. The transgenic monkeys showed less interaction with wild-type monkeys within the same group, and also a reduced interaction time when paired with other transgenic monkeys in social interaction tests. The cognitive functions of the transgenic monkeys were largely normal in the Wisconsin general test apparatus, although some showed signs of stereotypic cognitive behaviours. Notably, we succeeded in generating five F1 offspring of MECP2 transgenic monkeys by intracytoplasmic sperm injection with sperm from one F0 transgenic monkey, showing germline transmission and Mendelian segregation of several MECP2 transgenes in the F1 progeny. Moreover, F1 transgenic monkeys also showed reduced social interactions when tested in pairs, as compared to wild-type monkeys of similar age. Together, these results indicate the feasibility and reliability of using genetically engineered non-human primates to study brain disorders.
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Analysis of genomic integration sites of transgenes in F0 TG monkeys. (254 KB)
a, Genomic DNA were extracted from hair roots of WT and TG monkeys, digested and analysed by agarose gel. Radioactive probe labelled by 32P were prepared against the hMeCP2-2a-GFP transgene. Blots were transferred to membrane and hybridized with the probes. Images were acquired by exposing the blot to a phosphor-imager. Asterisk indicates target band. b, Flowchart of deep-sequencing-based methods for identifying genomic integration sites of lentiviral transgenes. Genomic integration sites of lentiviral transgenes are composed as three parts, endogenous genomic segments (black), LTRs (blue) and transgenes (red). Genomic DNA was sonicated, end-repaired and ligated to a Y-shaped adaptor, then subjected to two rounds of amplifications by the LTR1 + YP1 and LTR2 + YP2 primer sets. Illumina sequencing linkers were added onto segments and performed paired-end high-throughput sequencing. Target sequences containing LTR and endogenous genomic segments were collected and analysed. c, Comparison of copy numbers obtained from two methods among F0 TG monkeys. Red denotes copy numbers from AccuCopy (MECP2 and mCherry transgenes); blue denotes LTR insertion sites from deep-sequencing.
- Extended Data Figure 2: Physical parameters measured for monkeys. (334 KB)
Developmental changes in the body weight of 8 TG and 18 WT monkeys. a–g, Body weight, abdominal circumference, heart rate, respiratory rate, head circumference, body temperature and head–truck length were measured for 8 MECP2 TG and 18 WT monkeys. *P < 0.05 (Mann–Whitney U test). Error bars denote s.e.m.
- Extended Data Figure 3: Fatty acid measurements for TG and WT monkeys. (185 KB)
a, Blood samples collected at 18 months of age. b, Blood samples collected at 36 months of age. The blood levels of different forms of fatty acids were measured by mass spectrometry, with each bar represents results from three independent samples. C0, total fatty acid contents. All data are normalized to the average values of parallel blood samples from WT monkeys. *P < 0.05 (Student’s t-test). Error bars denote s.e.m.
- Extended Data Figure 4: Physical growth parameters measured for monkey T05 and transcriptome analysis of MECP2 transgenic monkey. (374 KB)
a–c, Body weight, head circumference and body temperature were measured for monkey T05. *P < 0.05 (Mann–Whitney U test), together with the average data from all other TG and WT monkey monitored. The monkey T05 died at 20 months of age. d, Volcano map for alterations in gene expression in the TG monkeys (T14, T05, T07 and T09), as compared to four WT monkeys. Red dots denote genes with a >2-fold change (FC) in expression (log2(FC(TG/WT)) > 1 or <−1). Blue dots denote genes with no significant change in expression (P > 0.05). e, Heat map representation of the selected genes involved in metabolic pathways and brain development. Gene expression is coded in pseudocolour scale (−0.14 to 0.14). Red denotes higher expression in TG monkeys; green denotes lower expression in TG monkeys, as compared to WT monkeys. Error bars denote s.e.m.
- Extended Data Figure 5: Anxiety responses in MECP2 TG monkeys. (147 KB)
a, Schematic illustration of the protocol of TAD test. b, Boxplots of the total numbers of grunt sounds made by WT and TG monkeys during the gaze period (‘step 4’ in the TAD test) at 36 months of age. c, Total grunts responses of wild-type and transgenic monkeys during TAD tests. d, Total vocal responses of wild-type and transgenic monkeys during TAD tests. *P < 0.05 (Student’s t-test). Ends of whiskers represent the minimum and maximum of data points. The line within box represents the median (odd numbers of data points) or second quartile (even number of data points). The bottom and top edge of the box represents the first and the third quartile, respectively.
- Extended Data Figure 6: Spectrograms of typical sounds produced by the monkey in the TAD test. (1,029 KB)
a–c, The power at different frequencies (ordinate) is colour-coded (red denotes higher power). Three typical sounds, grunt (a), coo (b) and scream (c), are shown.
- Extended Data Figure 7: Social interaction between monkeys from the same group (familiar pairing). (106 KB)
a–f, The average total time spent in sitting together during pairing in an isolated observation cage for each TG monkey (T04, T07, T08, T06, T09 and T11) with either a WT or a TG monkey was presented individually for six TG monkeys tested. T04 (a), T07 (b), T08 (c), T06 (d), T09 (e) and T11 (f). (See Supplementary Table 4c for grouping.) Each observation lasted 60 min daily for 5 days.
- Extended Data Figure 8: Schematic illustration of experimental procedures of WGTA test. (271 KB)
a, Black/white test. b, Boxplots of days required to pass the adaptation, discrimination and reversal steps in the black/white test for six WT and eight TG monkeys. Ends of whiskers represent the minimum and maximum of data points. The line within box represents the median (odd numbers of data points) or second quartile (even number of data points). The bottom and top edge of the box represents the first and the third quartile, respectively. c, Hamilton search test. d, Learning curves for the Hamilton forced set-breaking test after passing the black/white test (for six WT and seven TG monkeys). The difference between the two groups was at a significance level of P = 0.06 (Mann–Whitney U test). Error bars denote s.e.m.
- Extended Data Figure 9: Performance of WT and TG monkeys in learning set of WGTA test. (302 KB)
a, Learning set test. Correct rate of monkeys in the reward-shape association learning test plotted individually against trials, with data points represents average correct rates over 180 trials. b, WT monkeys. c, TG monkeys.
Extended Data Tables
- Video 1: Video 1:Example of normal motor behaviours (3.14 MB, Download)
- This video shows the representative normal motor behaviours for monkeys we examined.
- Video 2: Video 2: Example of repetitive motor behaviours (3.14 MB, Download)
- This video shows the typical repetitive motor behaviours for four F0 TG monkeys out of eight F0 TG monkeys we examined.
- Video 3: Video 3: Example of social interaction behaviours. (4.25 MB, Download)
- This video shows the representative social interaction behaviours of monkeys, such as sitting together, in natural community.
- Video 4: Video 4: Social Interaction behaviours in pairing experiments (393 KB, Download)
- This video shows the typical social behaviour, sitting together, in pairing experiments.
- Video 5: Video 5: Examples of learning behaviors of monkey in learning set of WGTA tests. (2.38 MB, Download)
- This video shows the example of learned behaviours for W09 out of five WT monkeys we examined.
- Video 6: Video 6. Examples of stereotypic behaviours of MECP2 F0 TG monkey in learning set of WGTA tests (2.31 MB, Download)
- This video shows the example of stereotypic responses of F0 TG monkeys (T09) out of seven F0 TG monkeys we examined.
- Supplementary Figures (3.4 MB)
This file contains the Western blots raw data for Figures 1d, e, f, h and 4c.
- Supplementary Tables (428 KB)
This file contains Supplementary Tables 1-7
- Supplementary Information (686 KB)
This zipped file contains Supplementary Audio files 1-3 comprising: (1) Grunt vocal responses of monkeys examined in TAD tests; (2) Coo vocal responses of monkeys examined in TAD tests; (3) Stream vocal responses of monkeys examined in TAD tests.