Th DpnI, MboI or BfuCI enzyme overnight. Agarose gel separation and Southern analysis was then performed as mentioned above.AcknowledgmentsWe would like to thank 
Dr Zainol Harun, consultant pathologist of Island Hospital Penang Malaysia, for assisting in identification and characterisation of the tumours.Plasmid Rescue ExperimentsStbl3 E. coli cells (Invitrogen, UK) were transformed by heatshock, using 15 mg DNA prepared by total cellular and tumour DNA isolation. DNA was concentrated using a Genomic DNA Clean and Concentrator kit (Zymo Research, USA) according to manufacturer’s instructions. Transformed colonies were selected on agar plates Pentagastrin web containing 30 mg/ml kanamycin. DNA was isolatedAuthor ContributionsConceived and designed the experiments: OA SPW. Performed the experiments: OA SPW KG. Analyzed the data: OA SPW. Contributed reagents/materials/analysis tools: OA SPW RPH. Wrote the paper: OA SPW RPH.S/MAR Vectors for In Vivo Tumour Modelling
Epithelial-mesenchymal transition (EMT) has been implicated as a means by which normal or transformed epithelial cells acquire the abilities to invade, resist apoptosis, and disseminate during development and cancer progression [1,2,3]. EMT, although not always the case, is generally considered as a prerequisite step during initial phase of metastasis. Multiple transcriptional factors, including Twist, Snail, and Slug, orchestrate the EMT and the migratory processes during embryogenesis. These factors have also been shown to promote cancer invasion and in metastasis in many experimental models of malignant tumors [4,5,6]. Growing evidence suggests that these transcription factors may regulate each other and control overlapping sets of target genes. The molecular mechanisms underlying the regulation of their interactions and expressions have not been well defined [7,8]. Recent understanding on EMT largely came from in vitro studies [9,10]. It’s difficult to validate whether carcinoma cells inhuman primary tumors have gone through an EMT in vivo. It is well-known that cells undergoing an EMT not only change their cellular characteristics to acquire motility and invasiveness but also develop new interactions with the extracellular environment. A hallmark of EMT is the loss of E-cadherin expression. However, some clinical observations showed that the majority of human breast carcinoma metastases express E-cadherin and maintain their epithelial morphology, suggesting that they have disseminated without switching to a mesenchymal phenotype or undergone mesenchymal-epithelial transition (MET) after metastatic growth [11,12]. Twist1 and Twist2 (dermo1), the basic helix-loop-helix (bHLH) transcriptional factor family, share more than 90 sequence homology and structural similarity at bHLH and C-teminal domains. They also overlap in temporal and spatial expression, and play critical roles in embryonic mesenchymal development [13]. A number of studies showed the important role of Twist1 in promoting cell survival, cell invasion and immigration [14,15], andHeterogeneous Twist2 Expression in Breast Cancersfacilitating tumor angiogenesis [16]. Both Twist1 and Twist2 are known to mediate EMT in human cancers [17]. Twist1 is a key regulator of metastasis. It has been shown that Twist1 promotes EMT through down-regulation of E-cadherin in subsets of sporadic invasive human lobular breast cancer [18], but little is known about the expression pattern of Twist2 [19,20]. Twist2 activates EMT programs and MedChemExpress SC-66 facilitates.Th DpnI, MboI or BfuCI enzyme overnight. Agarose gel separation and Southern analysis was then performed as mentioned above.AcknowledgmentsWe would like to thank Dr Zainol Harun, consultant pathologist of Island Hospital Penang Malaysia, for assisting in identification and characterisation of the tumours.Plasmid Rescue ExperimentsStbl3 E. coli cells (Invitrogen, UK) were transformed by heatshock, using 15 mg DNA prepared by total cellular and tumour DNA isolation. DNA was concentrated using a Genomic DNA Clean and Concentrator kit (Zymo Research, USA) according to manufacturer’s instructions. Transformed colonies were selected on agar plates containing 30 mg/ml kanamycin. DNA was isolatedAuthor ContributionsConceived and designed the experiments: OA SPW. Performed the experiments: OA SPW KG. Analyzed the data: OA SPW. Contributed reagents/materials/analysis tools: OA SPW RPH. Wrote the paper: OA SPW RPH.S/MAR Vectors for In Vivo Tumour Modelling
Epithelial-mesenchymal transition (EMT) has been implicated as a means by which normal or transformed epithelial cells acquire the abilities to invade, resist apoptosis, and disseminate during development and cancer progression [1,2,3]. EMT, although not always the case, is generally considered as a prerequisite step during initial phase of metastasis. Multiple transcriptional factors, including Twist, Snail, and Slug, orchestrate the EMT and the migratory processes during embryogenesis. These factors have also been shown to promote cancer invasion and in metastasis in many experimental models of malignant tumors [4,5,6]. Growing evidence suggests that these transcription factors may regulate each other and control overlapping sets of target genes. The molecular mechanisms underlying the regulation of their interactions and expressions have not been well defined [7,8]. Recent understanding on EMT largely came from in vitro studies [9,10]. It’s difficult to validate whether carcinoma cells inhuman primary tumors have gone through an EMT in vivo. It is well-known that cells undergoing an EMT not only change their cellular characteristics to acquire motility and invasiveness but also develop new interactions with the extracellular environment. A hallmark of EMT is the loss of E-cadherin expression. However, some clinical observations showed that the majority of human breast carcinoma metastases express E-cadherin and maintain their epithelial morphology, suggesting that they have disseminated without switching to a mesenchymal phenotype or undergone mesenchymal-epithelial transition (MET) after metastatic growth [11,12]. Twist1 and Twist2 (dermo1), the basic helix-loop-helix (bHLH) transcriptional factor family, share more than 90 sequence homology and structural similarity at bHLH and C-teminal domains. They also overlap in temporal and spatial expression, and play critical roles in embryonic mesenchymal development [13]. A number of studies showed the 
important role of Twist1 in promoting cell survival, cell invasion and immigration [14,15], andHeterogeneous Twist2 Expression in Breast Cancersfacilitating tumor angiogenesis [16]. Both Twist1 and Twist2 are known to mediate EMT in human cancers [17]. Twist1 is a key regulator of metastasis. It has been shown that Twist1 promotes EMT through down-regulation of E-cadherin in subsets of sporadic invasive human lobular breast cancer [18], but little is known about the expression pattern of Twist2 [19,20]. Twist2 activates EMT programs and facilitates.