A double fluorescence chimeric limb regeneration model reveals muscle fiber reconnection during axolotl limb regeneration / Modelo de regeneración quimérica de doble fluorescencia del miembro revela la reconexión de la fibra muscular durante la regeneración del miembro axolotl

SUMMARY: Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages.

RESUMEN: La regeneración del miembro de Axolotl es una característica fascinante que ha llamado la atención durante varias décadas. Nuestros estudios previos sobre la regeneración del miembro del Axolotl indicaron que las células satélite en los músculos remanentes se mueven distalmente hacia el blastema para regenerar nuevos músculos que están separados por una brecha de músculos remanentes. A partir de entonces, las fibras musculares regenerativas comienzan a reconectarse con las restantes. En este estudio, se aclaró la reconexión a nivel de fibra muscular individual para probar la hipótesis de que esta reconexión ocurre sincrónicamente entre los músculos involucrados. Se trasplantaron tres pares de blastemas EGFP+ en la etapa de yema media en tocones recién amputados de axolotls RFP+ en la misma posición del muslo para generar miembros posteriores regenerativos quiméricos de fluorescencia doble. Estos miembros regenerativos se cosecharon muy tarde mucho más allá de haber alcanzado la etapa de diferenciación tardía. Las imágenes de fluorescencia de estos miembros en secciones transversales revelaron que en la parte remanente proximal de la fibra muscular, la reconexión se produjo a un ritmo diferente entre los músculos. En el músculo grácil, la reconexión comenzó desde la periferia antes de completarse. Además, las fibras musculares RFP+ contribuyeron a la regeneración muscular en las partes regenerativas distales. Curiosamente, esta contribución de glóbulos rojos se limitó a los músculos superficiales ventrales de la pantorrilla. Este tipo de modelo de regeneración quimérica de doble fluorescencia del miembro puede ayudar a aumentar la comprensión del patrón de la regeneración del miembro del Axolotl en etapas tardías.

Immunogenicity of multi-epitope DNA vaccine of Mycobacterium tuberculosis and therapeutic effects of the vaccine combined with chemotherapy in a mouse model infected with multi-drug resistant Mycobacterium tuberculosis / 西安交通大学学报(医学版)

Objective To make a comparative study of the immunogenicity of multi-epitope DNA vaccine and BCG of Mycobacterium tuberculosis and therapeutic effects of the vaccines combined with chemotherapy in a mouse model infected with multi-drug resistant (MDR)Mycobacterium tuberculosis.Methods The BALB/c mice were randomly divided into Group A,Group B and Group C,which received subcutaneous immunization with PBS,BCG and multi-epitope DNA vaccine,respectively,once at weeks 1 ,3 and 5 .Specific IgG antibody,IL2 and IFN-γin mice serum were determined with ELISA after the final vaccination.At the same time,the splenic lymphocytes of mice were separated and the lymphocytes proliferation was determined by MTT method.To study the therapeutic effects of multi-epitope DNA vaccine,the mice were infected by intravenous injection in the tail vein with Mycobacterium tuberculosis clinical isolates that were resistant to isoniazid (INH)and rifampin (RFP).Four weeks later,the model mice were divided into Groups D,E and F.The mice in Group D and control group received saline injection. The mice in Group E were treated with RFP and INH.The mice in Group F were treated with multi-epitope DNA vaccine combined with INH and RFP for 10 weeks.The lungs,livers and spleens of mice were removed and weighed;the colony number of Mycobacterium tuberculosis in the lungs and spleens was counted.Results The antibody IgG,IL2 and IFN-γwere obviously higher in multi-epitope DNA vaccine group than in BCG group (P<0.05).Multi-epitope DNA vaccine combined with chemotherapy could obviously reduce the colony number of Mycobacterium tuberculosis in the lungs and spleens as well as indexes of the lungs,livers and spleens (P<0.05). Conclusion Mycobacterium tuberculosis Hsp70,Ag85A,and ESAT-6 multi-epitope DNA vaccine could induce strong specific immune response in mice that produced a high level of specific IgG antibody,IL2 and IFN-γ,specific lymphocyte proliferation,and significantly improve the efficacy of anti-tuberculosis drug resistant tuberculosis in mice.

Post-mortem re-cloning of a transgenic red fluorescent protein dog

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.