title: Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set.
abstract: BACKGROUND
  Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.
  
  
  RESULTS
  109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.
  
  
  CONCLUSIONS
  Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
authors.affiliation: Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kurome
authors.forename: Ludwig
authors.initials: L
authors.lastname: Geistlinger
authors.forename: Barbara
authors.initials: B
authors.lastname: Kessler
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
authors.forename: Nikolai
authors.initials: N
authors.lastname: Klymiuk
authors.forename: Annegret
authors.initials: A
authors.lastname: Wuensch
authors.forename: Anne
authors.initials: A
authors.lastname: Richter
authors.forename: Andrea
authors.initials: A
authors.lastname: Baehr
authors.forename: Katrin
authors.initials: K
authors.lastname: Kraehe
authors.forename: Katinka
authors.initials: K
authors.lastname: Burkhardt
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Flisikowski
authors.forename: Tatiana
authors.initials: T
authors.lastname: Flisikowska
authors.forename: Claudia
authors.initials: C
authors.lastname: Merkl
authors.forename: Martina
authors.initials: M
authors.lastname: Landmann
authors.forename: Marina
authors.initials: M
authors.lastname: Durkovic
authors.forename: Alexander
authors.initials: A
authors.lastname: Tschukes
authors.forename: Simone
authors.initials: S
authors.lastname: Kraner
authors.forename: Dirk
authors.initials: D
authors.lastname: Schindelhauer
authors.forename: Tobias
authors.initials: T
authors.lastname: Petri
authors.forename: Alexander
authors.initials: A
authors.lastname: Kind
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
authors.forename: Angelika
authors.initials: A
authors.lastname: Schnieke
authors.forename: Ralf
authors.initials: R
authors.lastname: Zimmer
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000502:physiology*; D001755:Blastocyst / Q000502:physiology; D003001:Cloning, Molecular; D003627:Data Interpretation, Statistical; D005260:Female; D055786:Gene Knockout Techniques; D008297:Male; D053652:Nuclear Transfer Techniques / Q000706:statistics & numerical data*; D011247:Pregnancy; D012621:Seasons; D050497:Stillbirth; D013552:Swine / Q000235:genetics / Q000502:physiology*