University of Veterinary Medicine Vienna - Research portal

Diagrammed Link to Homepage University of Veterinary Medicine, Vienna

Selected Publication:

Publication type: Diploma Thesis

Year: 2014

Author(s): Sauer, Mareike

Title: Genotypisierung der Keimzellen von Chimären zur Voraussage der Vererblichkeit induzierter Mutationen.

Other title: Identification of germnline competent chimaeras by spermatozoa genotyping

Source: Diplomarbeit, Vet. Med. Univ. Wien, pp. 42.


Advisor(s):

Rülicke Thomas

Reviewer(s):
Aurich Christine

Vetmed Research Units:
Institute of Laboratory Animal Science


Graduation date: 27.06.14


Abstract:
Due to the increased production of C57BL/6 derived ES cells for the generation of knockout mice, selecting germline competent chimaeras by coat colour chimaerism is no longer a reliable method, since even highly agouti C57BL/6 chimaeras can fail to transmit the manipulated allele. In genotyping sperm cell DNA, chimaeras with ES cell contribution to their germline, can be identified and subsequently used for matings. Chimaeras used in this study were mated with C57BL/6Tac mice. Mating results and the estimated degree of coat colour chimaerism was compared to the results obtained in the neomycin genotyping PCR. Sperm cell samples were collected either by separating mice during ejaculation and/or by transepidermal sperm cell aspiration. For sperm cell DNA extraction a precipitation method was used, as well as a binding column protocol. The extracted samples were used as a template for the genotyping PCR. All chimaeras, which had transmitted the gene of interest in matings showed a neo signal in PCR analysis. Chimaeras, which did not show a neo signal, also had not transmitted in matings. Consequently, chimaeras, not showing a signal for the induced mutation in their sperm cell samples, can be excluded from breeding. Identification of germline competent chimaeras by spermatozoa genotyping prior to breeding will lead to considerable savings in mouse numbers and resources.


© University of Veterinary Medicine ViennaHelp and Downloads