Fatty acid composition analysis
The hindquarter muscle samples from dead transgenic positive and negative calves were used for fatty acid analysis according to the procedures from Kang and Wang (2005). Briefly, tissue homogenate from muscle was mixed with 1 mL hexane and 1 mL 14 % BF3/ MeOH reagent in a glass methylationtube and evaporated with nitrogen. Then, the mixtures were incubated in a boiling water bath for 1 h. After cooled down to room temperature, the mixtures were blended with 1 mL H2O and centrifuged for 1 min. And then the upper hexane layer was reserved and concentrated under nitrogen. Fatty acid methyl esters were analyzed by gas chromatography (Agilent 7890A). The peak area was calculated by the peak height and width. Identification of components was determined by comparison of retention times with those of authentic standards (Sigma).
Results
Embryo transfer and production of mfat1 transgenic cattle
A total of 209 recipients cattle were synchronized before embryo transfer and the estrus synchronization rate were 68.1 and 53.8 % (Table 1). Ninety-foursynchronized recipients were transplanted with mfat1 transgenic embryos, and 20 calves were obtained by caesareansection.Threemfat1transgeniccattlestayed alive, and other seventeen cattle died after birth to 111 days (Table 2).
Anatomical analysis of the dead cloned cattle
Anatomical analysis of 17 dead cloned cattle showed that inflammation and hemorrhagic septicemia were the main causes of death (Table 2).
Mfat1 gene insertion analysis
Totally twenty cloned calves were obtained and 14 calves were transgenic positive identified by PCR (Figs. 2, 3). The PCR products were also verified by DNA sequencing and amino acid sequence alignment, whichwere consistent withtheaminoacid sequence of C. elegans fat1gene(data not shown).
Expression analysis of mfat1 gene
RT-PCR results showed that the mfat1 gene was expressed in liver, muscle, kidney and lung of the fat1