利用共分离和基因敲除技术分析小鼠大脑中金属硫蛋白基因表达与乙醇偏好的关系。

Analysis of metallothionein brain gene expression in relation to ethanol preference in mice using cosegregation and gene knockouts.

作者信息

Loney Kimberly D, Uddin Raihan K, Singh Shiva M

机构信息

Molecular Genetics Unit, Department of Biology and Division of Medical Genetics, University of Western Ontario, London, ON, Canada N6A 5B7.

出版信息

Alcohol Clin Exp Res. 2006 Jan;30(1):15-25. doi: 10.1111/j.1530-0277.2006.00015.x.

DOI:10.1111/j.1530-0277.2006.00015.x

PMID:16433728

Abstract

BACKGROUND

Metallothioneins (MTs) are ubiquitously expressed intracellular proteins that bind heavy metals and are involved in cytoprotection against several types of stress agents including chemicals, hormones, and oxidants. We have previously reported 1 isoform, MT-II, as a possible candidate gene for ethanol (EtOH) preference (EP) determination in mice.

METHODS

Semiquantitative RT-PCR was used to determine brain mRNA levels of MT-I and MT-III in 4 inbred mouse strains with variable EP. Following this, cosegregation of MT-II brain expression with EP was analyzed in F2 mice from 2 intercrosses (C57BL/6J x BALB/cJ and C57BL/6J x DBA/2J). Studies on MT-I/MT-II knockout (KO) mice were also undertaken to further explore this relationship.

RESULTS

Our results suggest that MT-I is responsive to EtOH, with no evidence of basal-level differences between strains. Conversely, MT-III shows no EtOH response, yet indicates a possible strain-specific feature with C57BL/6J having the lowest levels of brain MT-III. Metallothionein-II expression cosegregates with EP in F2 mice from a C57BL/6J (preferring) and DBA/2J (avoiding) intercross. Although F2 mice from a cross with C57BL/6J and BALB/cJ (avoiding) strains follow a similar pattern, the results are not statistically significant. Metallothionein-I/MT-II knockout (MT-KO) mice appear to have smaller litter sizes as well as higher weight compared with controls (129S1/SvImJ) and also show a slight increase in EP.

CONCLUSIONS

Metallothionein-II remains the primary candidate of the mouse MT gene family for involvement in EP. Its effect on EP appears to be dependent on the genetic background. Such conclusions are based on results from C57BL/6J, BALB/cJ, DBA/2J, and 129 inbred mouse strains. Evidence also points to shared neural pathways involved in weight gain and obesity. The complex interactions between MT-II, EP, and weight gain/obesity remain to be studied.

摘要

背景

金属硫蛋白（MTs）是细胞内普遍表达的蛋白质，可结合重金属，并参与对多种应激源（包括化学物质、激素和氧化剂）的细胞保护作用。我们之前曾报道过一种异构体MT-II，它可能是小鼠乙醇（EtOH）偏好（EP）测定的候选基因。

方法

采用半定量逆转录聚合酶链反应（RT-PCR）来测定4种具有不同乙醇偏好的近交系小鼠品系中MT-I和MT-III的脑mRNA水平。在此之后，对来自2个杂交组合（C57BL/6J×BALB/cJ和C57BL/6J×DBA/2J）的F2小鼠中MT-II脑表达与乙醇偏好的共分离情况进行了分析。还对MT-I/MT-II基因敲除（KO）小鼠进行了研究，以进一步探索这种关系。

结果

我们的结果表明，MT-I对乙醇有反应，各品系间无基础水平差异的证据。相反，MT-III未显示出乙醇反应，但表明可能存在品系特异性特征，C57BL/6J脑MT-III水平最低。在C57BL/6J（偏好）和DBA/2J（回避）杂交产生的F2小鼠中，金属硫蛋白-II的表达与乙醇偏好共分离。虽然C57BL/6J与BALB/cJ（回避）品系杂交产生的F2小鼠遵循类似模式，但结果无统计学意义。与对照（129S1/SvImJ）相比，金属硫蛋白-I/MT-II基因敲除（MT-KO）小鼠的窝仔数似乎更小，体重更高，并且乙醇偏好也略有增加。

结论

金属硫蛋白-II仍然是小鼠MT基因家族中参与乙醇偏好的主要候选基因。其对乙醇偏好的影响似乎取决于遗传背景。这些结论基于C57BL/6J、BALB/cJ、DBA/2J和129近交系小鼠品系的结果。证据还指向了体重增加和肥胖所涉及的共同神经通路。MT-II、乙醇偏好和体重增加/肥胖之间的复杂相互作用仍有待研究。