Establishment Of Plcd1 Transgenic Mouse

PLCD1, coded by Plcd1 gene, belongs to the subtypesδof Phospholipase C and plays an important role in phosphoinositide metabolism and Ca2+ balance in cells. Our goals were to creat Plcd1 transgenic mouse and further study the biological functions of Plcd1 in the animal level combined with snthr-1Bao mouse which had been obtained by ENU-induced mutagenesis in our lab and had been confirmed to be of a deletion of Plcd1. This experiment was carried out as two consecutive aspects: the construction of expression vector containing Plcd1 and zygotic microinjection for Plcd1 transgenic mice.Part I :Construction of pEF6/V5-His-Plcd1 Eukaryotic Expression VectorPurpose: To obtain the whole length of Plcd1 gene and construct eukaryotic expression vector which could express the fusion protein containing PLCD1. Methods: The whole encoding sequence of Plcd1 was amplified through RT-PCR from the total RNA isolated from testis of C57BL/6J mice. The recombinant cloning vector, pMD19-T-Plcd1, was constructed based on pMD19-T Vector and Plcd1 PCR products through conjunction catalyzed by DNA Ligase, transformation of competent cellls, blue-white selection, PCR detection and sequencing. Suitable restriction endonuclease cutting sites were introduced to the two sides of target DNA fragment by the primers and PCR methods based on the template of pMD19-T-Plcd1. Depurated PCR products and pEF6/V5-His-LacZ were used for construction of the eukaryotic expression vector of pEF6/V5-His-Plcd1 after a series of treatments such as restriction endonuclease digestion, conjunction, transformation, Amp selection, PCR detection and sequencing respectively. Then the recombinant expression vector of pEF6/V5-His-Plcd1 were transfected into L929 cells for transient expression and PLCD1 fusion protein were identified by immunofluorescence technology. Results: We cloned the 2271bp target DNA fragment of Plcd1 and constructed the recombinant cloning vector of pMD19-T-Plcd1. Restriction endonuclease cutting sites of SpeI and BstBI were successfully introduced to the target DNA franment of Plcd1, and the recombinant expression vector of pEF6/V5-His-Plcd1 were successfully obtained and confirmed through a series of steps mentioned above, PLCD1 fusion protein was finally detected in L929 cells by immunofluorescence technology after transfection. Conclusions: We successfully cloned the 2271bp target DNA fragment of Plcd1 and obtained the recombinant cloning vector of pMD19-T-Plcd1 and the expression vector of pEF6/V5-His-Plcd1 in succession, which successfully expressed the PLCD1 fusion protein.Part II :Establishment of Plcd1 Transgenic Mouse by microinjectionPurpose: To generate Plcd1 transgenic mouse by microinjection based on the recombinant expression vector of pEF6/V5-His-Plcd1. Methods: Linearized DNA fragmnent for microinjection were prepared by the restriction endonuclease digestion of pEF6/V5-His-Plcd1, followed by electrophoresis and purification. The donor and acceptor mice were treated to oestrus synchronization or superovulation at the same time and the fertilized ova were collected for microinjection. The survival zygotes after microinjection were reimplanted into the oviducts of pseudopregnant ICR female acceptors. Genomic DNA was extracted from the tail tips of pups to check whether the transgenic construction were integrated into the mouse genome or not, and then positive transgenic animals were identified by PCR detection. Results: We got the DNA fragmnent for microinjection containing hEF-1αpromoter, the target DNA fragment of Plcd1, V5, His-tag and BGH from pEF6/V5-His-Plcd1 digested by restriction endonuclease NruI and AseI. In the study, only 393 of 681 zygotes(57.7%) survived the microinjection of transgenic construction and these survival zygotes were reimplanted into the oviducts of 18 pseudopregnant ICR female mice. 13 out of 18 ICR female mice were pregnant and 82 pups were born at last. PCR analysis indicated that 15 out of 82 pups (18.3%)were integrated with exogenous Plcd1. Conclusions: 15 Plcd1 transgenic founder mice were preliminary confirmed by PCR detection. The Plcd1 transgenic founder mice provide a new animal model for studying the functions of Plcd1 gene.