Preliminary Characterization of a Leptin Receptor Knockout Rat Created by CRISPR/Cas9 System

Leptin receptor, which is encoded by the diabetes (db) gene and is highly expressed in the choroid plexus, regulatesenergy homeostasis, the balance between food intake and energy expenditure, fertility and bone mass. Here, using CRISPR/Cas9 technology, we created the leptin receptor knockout rat. Homozygous leptin receptor null rats are characterized by obesity, hyperphagia, hyperglycemia, glucose intolerance, hyperinsulinemia and dyslipidemia. Due to long-term poor glycemic control, the leptin receptor knockout rats also develop some diabetic complications such as pancreatic, hepatic and renal lesions. In addition, the leptin receptor knockout rats show a significant decrease in bone volume and bone mineral density of the femur compared with their wild-type littermates. Our model has rescued some deficiency of the existing rodent models, such as the transient hyperglycemia of db/db mice in the C57BL/6J genetic background and the delayed onset of glucose intolerance in the Zucker rats, and it is proven to be a useful animal model for biomedical and pharmacological research on obesity and diabetes.

Leptin receptor knockout induced obesity and hyperphagia. The body weight was measured from 1 to 8 months of age. The male Lepr −/− rats emerged with severe early-onset obesity as early as 1 month of age and were approximately 60% heavier at 8 months of age compared with their wild-type (WT) littermates ( Fig. 2A,B, n = 12, P = 0.002). The female Lepr −/− rats presented more severe obesity than male rats and were approximately160% heavier at 8 months of age (Fig. 2C, n = 15, P = 0.02). The increased body weight of Lepr −/− rats was associated with significantly elevated daily food consumption in both genders (Fig. 2D,E).

Figure 1. Generation of Leptin receptor knockout rat using CRISPR/Cas9 system. (A) Target loci of
Lepr were amplified using genomic DNA templates from founders. M: DNA molecular weight marker DL2000; WT: Template DNA was replaced with wild-type genomic DNA; 1-8: Founder rats generated by microinjection. (B) PCR products of the targeted fragment in the Lepr in rats were sequenced. The protospacer adjacent motif (PAM) sequence was underlined and highlighted in green; the targeting sites were red; the insertions were purple, lower case; insertions (+ ) or deletions (− ) were shown to the right of each allele. The E3, E4 and E5 represents exon 3, exon 4 and exon 5 of Lepr respectively. (C) Protein level of LEPR in the liver tissues of WT littermates and Lepr −/− rats were detected by western blot, using β -actin as normalization.
The male Lepr −/− rats emerged with higher fasting glucose level at 4 months of age and this hyperglycemia continued to 8 months of age (Fig. 3A). Significantly higher random glucose levels occurred as early as 2 months of age in male Lepr −/− rats and reached peak glucose at 4 months of age, which increased 1.64-fold compared with those of their WT littermates (Fig. 3C, n = 12, P = 0.02). Random hyperglycemia also continued to 8 months of age. However, persistent hyperglycemia was not observed in the female Lepr −/− rats (Fig. 3B,D).
According to the above-described glucose levels, we chose 2-, 4-and 8-month old rats on which to performa glucose tolerance test. After the glucose loading, the Lepr −/− rats showed rapid and remarkable elevation of serum glucose levels, whereas serum glucose elevation was relatively slow and brief in their WT littermates. Glucose intolerance appeared in maleLepr −/− rats at 2 months of age and deteriorated with aging. In particular, at 8 months of age the peak glucose level reached 22.2 mmol/L and the glucose level was sustained at 16.3 mmol/L at 120 min after glucose loading (Fig. 3E, n = 8, P = 0.002). In female Lepr −/− rats, glucose intolerance only was observed at 4 months of age (Fig. 3F).
To further evaluate glucose homeostasis in Lepr −/− rats, serum insulin level were measured in a glucose tolerance test. The Lepr −/− rats showed significant hyperinsulinemia at baseline and presented a dramatic increase in serum insulin levels at 2, 4 and 8 months of age in both genders after the glucose loading (Fig. 3G,H).
Leptin receptor knockout induced dyslipidemia. Diabetes-associated lipid metabolism parameters were measured at 2, 4 and 8 months of age in both genders. The Lepr −/− rats showed similar lipid metabolism characteristics in both genders, so we merged the parameters of males and females (Table 1). Circulating triglycerides, total cholesterol and high density lipoprotein were all significantly increased in Lepr −/− rats compared with those of their WT littermates at 2, 4 and 8 months of age. A diabetic

Discussion
In the past few decades, obesity and obesity-related disease, such as type 2 diabetes, have been shown to be directly related to increased mortality and reduced life expectancy 20 . Human type 2 diabetes is a complex heterogeneous disease; it is clinically characterized by obesity, overt hyperglycemia, dyslipidemia and glucose intolerance 21 . Moreover, long-term poor glycemic control in diabetic patients leads to the development of microvascular and macrovascular complications 22,23 . Patients with type 2 diabetes also present with a higher potential for falls and risk for fracture than nondiabetic individuals 24,25 .
Various animal models of type 2 diabetes have been established to study human type 2 diabetes. However,these models do not develop the full phenotype of type 2 diabetes. Thus, another animal model, in particular a rat model, due to its physiology advantages compared with mice, should be helpful for Lepr's clinical applications.
Here, we created theLepr knockout rats (named as Lepr tm1Ilas in our laboratory Rat Resource website: http://123.1.153.158/portal/root/website_yky) using CRISPR/Cas9 technology.The phenotypes of thedb/db mice, the Zucker rats and theLepr −/− rats was summarized in Table 2. Our two gRNAs targeting the fourth exon of the Lepr gene, induced a 298-bp deletion and a 4-bp insertion and resulted TGA termination codon prematurely, which led to the absence of LEPR in the Lepr −/− rats. However, the Zucker rat model had a spontaneous missense mutation in the Lepr gene, which does not affect the expression level of LEPR 26 . The differences in LEPR expression resulted in disparate phenotypes between our Lepr −/− rats and the Zucker rats. The Lepr −/− rats are obese and develop mild random hyperglycemia, hyperinsulinemia and dyslipidemia as early as 8 weeks of age. Whereas, the Zucker rats do not present with typical high blood glucose levels 10 , and they develop glucose intolerance and insulin resistance within 12 weeks of age in a mixed genetic background (a cross between Merck 3M and Sherman rats) 27 , and demonstrate a delayed onset of glucose intolerance to 21-23 weeks when bred into the SD background 28  with the Zucker rats, our Lepr −/− rats demonstrated a significant decrease in BV/TV compared with their WT littermates 29 . Leptin has been found to restrain corticotropic releasing hormone and to stimulate gonadotropin releasing hormone release from the hypothalamus 30 . Leptin-deficiency or Lepr-deficiency obese animals are both hypercortisolic and hypogonadal 3 . Increased corticosteroid production and decreased estrogen presence favor an increase in osteoclast number and subsequent increase in bone resorption, which may predominate in the Lepr −/− rats to increase the bone loss. Another commonly used animal model of type 2 diabetes is db/db mice, which are characterized by hyperphagia, morbid obesity and extreme insulin resistance 5,31 . The hyperglycemia of db/db mice depends on the background strain. For example, db/dbmice present with transient hyperglycemia in a C57BL/6J genetic background; although db/db mice in C57BL/KSJ genetic background exhibit uncontrollable hyperglycemia, they could only survive to 10 months of age 32 . Our Lepr −/− rat presents with mild hyperglycemia as early as 1 month of age and this higher levels continued to 8 months of age. The chronic hyperglycemia of the Lepr −/− rat demonstrates the advantage of long-term observation on the development of diabetes and diabetes-related complications. The db/db mice and the Lepr −/− rat can possible complement each other in research on the development of diabetes.
In conclusion, our initial characterization shows that knockout of the Lepr gene in SD rats leads to severe obesity, hyperphagia, glucose intolerance, hyperinsulinemia, dyslipidemia, decreased bone mineral   density and partial diabetes complications. Our model compensates for some deficiencies of the existing rodent models, especially with respect to chronic hyperglycemia, and it is proven to be a usefulanimal model for obesity and diabetes research.

Methods
The use of animals and all experimental protocol were approved by the Animal Care and Use Committees of The Institute of Laboratory Animal Science of Peking Union Medical College (ILAS-GC-2010-044), including the establishment of the Leprknockout rats, fasting and random glucose test, glucose tolerance test, serum insulin level test, serum biochemistry test, histological analysis and microcomputedtomography analysis. And all the methods were carried out in accordance with the approved guidelines mentioned above.
Animals. The Lepr knockout rats were generated by CRISPR/Cas9 as described previously 33 . In brief, we designed two pairs ofsynthesized oligonucleotides for gRNA targeting on the exon 4 of Lepr, TAGGCAAATCATCTATAACTTC and AAACGAAGTTATAGATGATTTG; TAGGCTGAAAGCTGTCTTTCAG and AAACCTGAAAGACAGCTTTCAG, which were annealed and cloned into the pUC57-gRNA expression vector ( Protein Extraction and Western Blot Analysis. The rats were euthanized and total protein lysates from the rat liver tissues were prepared as previously described 34 . After SDS-PAGE and transfer of the bands to nitrocellulose (Millipore), the membranes were incubated overnight with antibodies against LEPR (Santa Cruz, sc-8325). After incubation with the appropriate secondary antibody for 1h at room temperature, antibody binding was detected with an HRP-conjugated immunoglobulin G (Santa Cruz) using a chemiluminescence detection system (Santa Cruz). For quantitative analysis, the LEPR level was normalized to β -actin.
Body Weight and Food Consumption. WT littermates and Lepr −/− rats of both genders were weighed every month from 1 to 8 months of age. WT littermates and Lepr −/− rats were provided with standard food and water libitum. Food was weighed, and the average daily intake was calculated from 1 to 8 months of age.
Fasting and Random Blood Glucose. WT littermates and Lepr −/− rats of both genders were fasted overnight (14 h) but given water libitum. Blood was collected by tail vein puncture and blood glucose was analyzed by a One Touch Ultra glucometer (YZB/USA 6891). Random blood glucose measurement was performed at 9:00 a.m. over 8 month in both genders.
Glucose Tolerance Test and Serum Insulin Level. WT littermates and Lepr −/− rats of both genders were fasted overnight (14 h) but given water libitum. On the day of the test, the rats were weighed, and blood was collected by tail vein puncture. Blood glucose was analyzed by a One Touch Ultra glucometer (YZB/USA 6891). After a baseline glucose concentration was obtained, the rats were injected intraperitoneally with D-glucose at 1 g/kg body weight. Blood glucose levels were sampled from the tail at 30, 60, 90 and 120 min after injection. Meanwhile, 100 μ l of blood was collected for serum insulin level test using rat/mouse insulin ELISA kits (Millipore). Serum Biochemistry. WT littermates and Lepr −/− rats of both genders were fasted overnight (14 h) but given water libitum. Blood was collected by tail vein puncture. Whole blood was centrifuged at 3000 g for 10 min at 4 °C to obtain the serum and prepared for serum total cholesterol (CHO), triglycerides Scientific RepoRts | 5:15942 | DOi: 10.1038/srep15942 (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) detection using a HITACHI 7100 Automatic Analyzer.
Histological Analysis. For light microscopy, the rats were euthanized, and the pancreas, liver, kidney and abdominal adipose tissue were fixed in 4% formaldehyde and mounted in paraffin blocks. The sections were stained with haematoxylin and eosin (H&E) and analyzed using the Aperio Image Scope v8.2.5 software. The sections were analyzed by an observer blinded to the rat genotypes.
Microcomputed Tomography (μCT) Analysis. WT littermates and Lepr −/− rats 8 months of age were euthanizedand their femurs were dissected. Measurements of trabecular architecture were performed on the distal femur cleared of all soft tissue using Siemens INVEON LG CT. After an initial scout scan, a total of 100 slices with an increment of 10 μ m were obtained on each bone sample, starting 1.0 mm below the growth plate. The area for analysis was outlined within the trabecular compartment, excluding the cortical and subcortical bone. Every 5 sections were outlined, and the intermediate sections wereinterpolated with the contouring algorithm to create a volume of interest. Segmentation values used for analysis were determined using Inveon Research Workplace. A three-dimensional (3-D) analysis was performed to determine BV/TV, trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). A two-dimensional (2-D) analysis was performed to determine bone mineral density (BMD). The mean cortical thickness (Ct.Th) was determined by distance measurements at 4 different points on the cortical slice.
Statistical Analysis. The data were analyzed by Two Independent-Samples non-parametric test. The data were expressed as the means ± SEMs from individual experiments. The differences were considered significant at P < 0.05.