Serum CA19-9 as a marker of circulating tumor cells in first reflux blood of colorectal cancer patients.

Circulating tumor cells (CTCs) are used for metastasis surveillance in cancer patients, but low detection rates limit their use in colorectal cancer (CRC). We investigated the distribution of CTCs in peripheral and portal blood of CRC patients, and analyzed the relationship between serum tumor CEA/CA19-9 markers and CTCs blood levels. CTC levels detected in first reflux/portal vein blood were higher than in peripheral blood, and liver reduced CTCs amount. CTCs-positive patients had increased serum CEA and CA 19-9 levels, and the CEA and CA 19-9 levels correlated with the CTCs levels. Even in non-metastatic CRC patients with barely detectable CTCs in peripheral blood, serum CA 19-9 levels correlated with the CTC levels in first reflux/portal vein blood. These results demonstrate that CTC detection in the first reflux vein/portal vein blood is more sensitive than in peripheral blood, suggesting that clinical diagnosis using the CellSearch System should be based on the CTC detection in first reflux vein blood due to the high detection rates. In addition, our results indicate that serum CA 19-9 levels may serve as a diagnostic marker for further evaluation of CTC levels in portal blood.

[Isolation of new Zinc finger genes through cDNA library screening combined with RACE].

Most of the important functionally proteins contain the corresponding function domains that consist of conserved amino acid sequences. The study provided a method to identify novel genes that encode proteins containing important functionally domains with conserved sequences. First, primers were designed according to the sequence of the cDNA library vector and the ESTs that have been obtained by reverse PCR and degenerate primers encoding Zinc finger domain. The cDNA library DNA was used as template for PCR amplification. The amplified fragment that contains nonhomologous sequences of the cDNA was inserted into pGEM-T easy vector. The fragment was recovered and used as a probe for screening the cDNA library. Several cDNAs with full length that encode proteins with Zinc finger domain and represent the original ESTs have been successfully cloned from a human bone marrow cDNA library. This strategy can also be used in screening genes that encode proteins containing differential function domains with conserved sequences.

[Sodium chloride step gradients centrifuge to fractionate foreign DNA fragment for microinjection].

DNA microinjection is the most popular and reliable method of producing transgenic animals. The purity of foreign DNA plays an important role for the success of microinjection. In this study, we introduced the use of sodium chloride step gradients in fractionating foreign DNA fragment for microinjection. The data demonstrated that,compared with the conventional agarose gel extraction method, NaCl purification scheme of toreign DNA could improve the treated embryo survival and foreign DNA intergration rate markedly.

T to C substitution at -175 or -173 of the gamma-globin promoter affects GATA-1 and Oct-1 binding in vitro differently but can independently reproduce the hereditary persistence of fetal hemoglobin phenotype in transgenic mice.

The T to C substitution at position -175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma-to beta-globin gene with the natural chromosome arrangement but with the -175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the mu'LCRAgamma(-173)psibetadeltabeta construct, in which the -175 T to C Agamma gene was substituted with the -173 T to C Agamma gene. In vitro experiments proved that the -175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the -173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the -175 mutation or the -173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences gamma-globin gene expression in normal adult erythrocytes. Both the -173 and -175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the gamma-globin gene in adult erythrocytes.