PMID- 11139228
OWN - NLM
STAT- MEDLINE
DCOM- 20010517
LR  - 20191210
IS  - 1040-452X (Print)
IS  - 1040-452X (Linking)
VI  - 58
IP  - 2
DP  - 2001 Feb
TI  - Assessment of the proportion of transgene-bearing sperm by fluorescence in situ 
      hybridization: a novel approach for the detection of germline mosaicism in 
      transgenic male founders.
PG  - 166-72
AB  - Genetic mosaicism is frequent among transgenic animals produced by pronuclear 
      microinjection. A successful method for the screening of founder animals for 
      germline mosaicism prior to mating would greatly reduce the costs associated with 
      the propagation of the transgenic lines, and improve the efficiency of transgenic 
      livestock production. With this aim, we have devised a simple method to detect 
      integrated transgenes in individual spermatozoa using fluorescence in situ 
      hybridization (FISH). The experiments reported here were undertaken to 
      investigate the efficiency of this FISH-based approach to accurately evaluate the 
      proportion of transgene-bearing sperm and to be applied for the detection of 
      potential germline mosaics. Sperm samples from mice homozygous and hemizygous for 
      a beta-lactoglobulin transgene were analyzed in a first set of experiments. A 
      high hybridization efficiency was achieved, and the proportions of 
      transgene-positive sperm cells in both homozygous (94.8-98.2%) and hemizygous 
      (49.8-51.9%) animals were close to the expected frequencies (100 and 50%, 
      respectively). To evaluate the sensitivity of the assay more directly, simulated 
      mosaic samples with 5, 10, 15, 20 and 40% of transgene-bearing spermatozoa were 
      then prepared and analyzed by FISH. Significant differences in the frequency of 
      transgene-positive sperm were observed between all mosaic samples, indicating 
      that even small deviations (5%) from the expected 50% transgene transmission rate 
      in a founder animal could be reliably detected with our assay. Therefore, the 
      method proposed represents a novel approach for the identification of germline 
      mosaic founder males in livestock transgenic projects and a much more economic 
      and faster alternative to breeding.
FAU - Ibanez, E
AU  - Ibanez E
AD  - Unitat de Biologia Cel.lular, Facultat de Ciencies, Departament de Biologia 
      Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 
      Bellaterra, Spain. Elena.Ibanez@uab.es
FAU - Molist, J
AU  - Molist J
FAU - Vidal, F
AU  - Vidal F
FAU - Egozcue, J
AU  - Egozcue J
FAU - Santalo, J
AU  - Santalo J
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Mol Reprod Dev
JT  - Molecular reproduction and development
JID - 8903333
RN  - 0 (Lactoglobulins)
SB  - IM
MH  - Animals
MH  - Founder Effect
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Lactoglobulins/*genetics
MH  - Male
MH  - Mice
MH  - Mice, Inbred C57BL
MH  - Mice, Transgenic
MH  - Mosaicism/*genetics
MH  - Sensitivity and Specificity
MH  - Spermatozoa/*metabolism
MH  - *Transgenes
EDAT- 2001/01/04 11:00
MHDA- 2001/05/18 10:01
CRDT- 2001/01/04 11:00
PHST- 2001/01/04 11:00 [pubmed]
PHST- 2001/05/18 10:01 [medline]
PHST- 2001/01/04 11:00 [entrez]
AID - 10.1002/1098-2795(200102)58:2<166::AID-MRD5>3.0.CO;2-Y [pii]
AID - 10.1002/1098-2795(200102)58:2<166::AID-MRD5>3.0.CO;2-Y [doi]
PST - ppublish
SO  - Mol Reprod Dev. 2001 Feb;58(2):166-72. doi: 
      10.1002/1098-2795(200102)58:2<166::AID-MRD5>3.0.CO;2-Y.