Irregular patterns of transgene silencing in allohexaploid oat

An irregular pattern of transgene silencing was revealed in expression and inheritance studies conducted over multiple generations following transgene introduction by microprojectile bombardment of allohexaploid cultivated oat (Avena sativa L.). Expression of two transgenes, bar and uidA, delivered on the same plasmid was investigated in 23 transgenic oat lines. Twenty-one transgenic lines, each derived from an independently selected transformed tissue culture, showed expression of both bar and uidA while two lines expressed only bar. The relationship of the transgenic phenotypes to the presence of the transgenes in the study was determined using (1) phenotypic scoring combined with Southern blot analyses of progeny, (2) coexpression of the two transgenic phenotypes since the two transgenes always cosegregated, and (3) reactivation of a transgenic phenotype in self-pollinated progenies of transgenic plants that did not exhibit a transgenic phenotype. Transgene silencing was observed in 19 of the 23 transgenic lines and resulted in distorted segregation of transgenic phenotypes in 10 lines. Silencing and inheritance distortions were irregular and unpredictable. They were often reversible in a subsequent generation of self-pollinated progeny and abnormally segregating progenies were as likely to trace back to parents that exhibited normal segregation in a previous generation as to parents showing segregation distortions. Possible causes of the irregular patterns of transgene silencing are discussed.     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.     

Gene silencing results in instability of antibody production in transgenic plants

The stability of antibody and F_ab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed. Received: 27 July 1998 / Accepted: 12 October 1998     

Stability of transgene methylation patterns in mice: Position effects,strain specificity and cellular mosaicism

The methylation status of a transgene, which carried the adenovirus type 2 E2A late promoter linked to the chloramphenicol acetyltransferase gene, was studied in three transgenic mouse lines (5–8, 7–1 and 8–1). These lines were analysed over a large number of offspring generations beyond the founder animal. In mating experiments, the influence of the parent-of-origin and strain-specific backgrounds on the transgene methylation patterns were assessed and found to have no effect on the pre-established methylation patterns in mouse lines 5–8 and 8–1. The founder animal 7–1 carried two groups of a total of ten transgenes, which were located on two different chromosomes. These arrays of transgenes could be segregated into separate mouse lines 7-1A and 7-1B. The transgenes of 7-1A animals exhibited cellular mosaic methylation, patterns that were demethylated in approximately 10% of the offspring in a mixed genetic background. Upon further transmission of these transgenes in a mixed genetic background, the grandparental methylation patterns were reestablished in most progeny. Mating to inbred DBA/2 mice resulted in maintenance of the demethylated pattern or in further demethylation of the transgenes in approximately 50% of the offspring. In contrast, an equal number of transgenic siblings from matings to C57BL/6 mice showed a return to the original methylation pattern. The mosaic methylation status of this locus was apparently controlled by mouse-strain-specific factors. The methylation patterns of the 7-1B transgenes were not cellular mosaic and remained stable in all offspring, as with lines 5–8 and 8–1. Hence, the strain-dependent and cellular mosaic transgene methylation patterns of 7-1A animals were probably a consequence of the chromosomal integration site of the transgenes (position effect).     

Genetic analysis and epigenetic silencing of At4CL1 and At4CL2 expression in transgenic Arabidopsis

4-coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. Transgenic Arabidopsis plants containing the At4CL1 or At4CL2 promoter fused to the beta-glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation of At4CL and other phenylpropanoid-specific genes, we generated ethyl methyl sulfate mutagenized populations of At4CL1::GUS and At4CL2::GUS transgenic lines and screened approximately 16,000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs enhanced the GUS-silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in the At4CL1::GUS and At4CL2::GUS silenced lines. While silencing in the seedlings of the At4CL1::GUS lines was root specific in seedlings, it affected all organs in the At4CL2::GUS lines. Also, At4CL1::GUS transgene silencing was confined to the transgene but At4CL2::GUS silencing extended to the endogenous At4CL2 gene. Organ-specific silencing of the At4CL1::GUS transgene cannot be explained by current models in the literature.